Enhanced disruption of Candida utilis using enzymatic pretreatment and high-pressure homogenization

The enhancement of the overall disruption of a native strain of Candida utilis (ATCC 9226) was studied using a combination of two methods, namely, pretreatment in the form of partial enzymatic lysis by Zymolyase followed by mechanical disruption in a Microfluidizer high-pressure homogenizer. The cells were grown in both batch and continuous cultures to examine the effect of specific growth rate on disruption. Cell suspensions ranging in concentration from 7 to 120 g DW/L were disrupted with and without enzymatic pretreatment. For yeast grown in batch culture, final total disruption obtained using the combined protocol approached 95% with four passes at a pressure of 95 MPa, as compared with only 65% disruption using only mechanical homogenization. A modified model was developed to predict the fraction disrupted by the enzymatic pretreatment-mechanical homogenization two-stage process. Predicted disruptions agreed favorably with experimental observations (maximum deviation of 20%) over a wide range of operating conditions. (c) 1994 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     

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.     

Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers

The protein releases, the particle size distribution and the viscosity of disrupted E. coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M-8TA and Microfluidizer M-110 were determined. The effects of these parameters on separation of the cell debris from the protein solution by centrifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably different physical properties of the cell disintegrates which influences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disruption in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, increasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high-pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disintegrate both in filtration and in centrifugation.     

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from <Emphasis Type="Italic">Escherichia coli</Emphasis>

A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps.     

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.     

Pyridine nucleotide transhydrogenations in yeast

Though previously described as very low or absent in yeast, we find significant pyridine nucleotide transhydrogenation (NADPH + acetyl pyridine-NAD+----NADP+ + acetyl pyridine-NADH) activity in yeast extracts when assayed at pH 8-9, and describe here the subcellular distribution and separation of the various molecular forms contributing to the total activity in two yeast species. Gentle subcellular fractionation reveals transhydrogenase activity only in the cytosolic fraction of both Saccharomyces cerevisiae and Candida utilis while intact mitochondria and microsomes are without activity. On sucrose gradient centrifugation, this soluble cytosolic activity proves to be primarily in a high-molecular-weight (greater than 10(6)) band which has salmon-colored fluorescence on uv illumination. Sonication of the particulate subcellular fractions solubilizes substantial transhydrogenase activity from mitochondria of C. utilis (but not from S. cerevisiae) which on sucrose gradients consists of both high (greater than 10(6))- and low-molecular-weight active fractions, each with yellow-green fluorescence. Ammonium sulfate fractionation and sucrose gradient centrifugation of protein solubilized from whole yeast of both species by vigorous homogenization with glass beads confirms the presence and fluorescence of these various molecular weight forms. The relationship of these activities to other enzymatic activities (especially the mitochondrial external NADH dehydrogenase) is discussed.     

Disruption of a filamentous fungal organism (N. sitophila) using a bead mill of novel design II. Increased recovery of cellulases

A native strain of Neurospora sitophila was disrupted using enzymatic pretreatment combined with mechanical disruption in order to facilitate recovery of constitutive cellulases. Exceptional disruption (approaching 100%) was achieved when the enzymatic pretreatment protocol was used prior to mechanical disruption at a low rotor speed via a new bead mill (the Annu Mill). Further, increased recovery of cellulases (ca. two-fold increases in cellulase activity per unit biomass) appears attainable when this disruption protocol is employed. The enzyme preparation employed was Zymolyase, which lyses the walls of viable fungi. Combined disruption of the mycelial biomass appears to provide a secondary source of cellulases from Neurospora sitophila in addition to the extracellular primary source derived from the filtered (unprocessed) fermentation broth.Nomenclature CMCase carboxymethyl cellulase - FPase filter paper'ase - IU international unit (mol liberated hydrolysis product/min.) - N number of passes through the bead mill (–) - R total fraction of cells disrupted (–) - Ro fraction of cells disrupted after enzymatic pretreatment alone (–) - X cell concentration (dry weight) (gDW/L) Abbreviations DW dry weight     

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.     

Significance of location of enzymes on their release during microbial cell disruption.

The release kinetics of the enzyme invertase and alcohol dehydrogenase from yeast and penicillin acylase from E. coli during disruption using various techniques has been investigated. The disruption techniques used were sonication, high-pressure homogenization, and hydrodynamic cavitation. The first-order-release kinetics was applied for the determination of release rate of these enzymes and total soluble proteins. Location factor (LF) values were calculated using these release rates. The location of the enzymes as given by the values of location factor coincided well with those reported in the literature. Varying values of location factor for the same enzyme by different disruption techniques gave some indications about the selectivity of release of a target enzyme by different disruption techniques. Varying values of location factor for the same enzyme with the use of a particular equipment or disruption technique at different conditions reveals the degree to which the cell is disrupted. Few plausible applications of this location factor concept have been predicted and these speculations have been examined. This location factor concept has been used for monitoring the heat-induced translocation of ADH and location of penicillin acylase during the growth period of E. coli cells.     

Release of aminopeptidase from Lactobacillus casei sp. casei by cell disruption in a Microfluidizer

Cell disruption in a Microfluidizer was a function of both operating pressure and number of passes. The operating pressure had greater effect on the disruption than did the number of passes as indicated by the magnitude of the constants from the cell disruption equation. Protein release correlated with aminopeptidase release by Lactobacillus casei sp. casei. The optimum operating pressure for enzyme extraction was 76 MPa with loss of enzyme activity about 15 to 20%.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Combination of cell disruption technologies for lipid recovery from dry and wet biomass of Yarrowia lipolytica and using green solvents

This work aims to investigate the efficiency of bead milling combined or not with high-pressure homogenization on the cell disruption and lipid recovery from Yarrowia lipolytica oleaginous yeast. First, a simulation study involving the use of the Hansen solubility parameters’ approach was performed in order to identify, among 41 conventional and “green” solvents, the most promising ones that are able to replace n-hexane for lipid recovery from Y. lipolytica biomass. The results obtained showed that the pre-treatment involving both high-pressure homogenization and bead milling applied sequentially was more performant than that involving bead milling alone. In addition, bead-milling parameters were optimized showing an optimal bead size of 4.9 mm and a processing time of 30 s. Among the tested solvents, isoamyl acetate was selected as the most appropriate “green” solvent, maximizing the lipid extraction, compared to n-hexane. Despite the better performance of the dry route compared to the wet one, promising results were obtained towards 1) minimizing the energy consumed and 2) replacing n-hexane by “green” solvents for lipid recovery from Y. lipolytica yeast.     

A microscale bacterial cell disruption technique as first step for automated and miniaturized process development

Cell disruption is crucial during recovery of biopharmaceuticals overexpressed in E. coli, which tend to be produced intracellularly as insoluble inclusion bodies. Miniaturized high-throughput systems can accelerate the laborious downstream protocol for such biopharmaceuticals and enable integrated process-development. A fast and robust cell disruption method reflecting the protein and impurity profile of homogenates obtained by large-scale methods is required for such an approach. We established a miniaturized bead mill for parallel mechanical cell disruption at the microscale. Its total protein and impurity release, protein pattern, and particle size distribution were compared to results from microscale enzymatic digestion and referred to laboratory-scale high-pressure homogenization. Bead mill disruption led to equivalent protein and impurity release as well as to the same particle size profile as the large-scale reference. In contrast, lysates obtained by enzymatic digestion contained only 30–47% of overall protein, 17% of dsDNA, and 7–10% of endotoxin compared to those obtained by high-pressure homogenization; also larger debris was present in lysates after enzymatic digestion. The established method is fast, efficient, robust and comparable to current large-scale standards, allowing for parallelization of experiments. Thus, it is the method of choice for rapid integrated process development at the microscale.     

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.     

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.     

The role of glutathione in amino-acid absorption. Lack of correlation between glutathione turnover and amino-acid absorption by the yeast Candida utilis.

The rate of degradation of glutathione has been determined in the yeast Candida utilis by using a method that minimizes the effect of amino-acid recycling. When yeast are grown in amino-acid-free medium, the half-life of glutathione was found to be 230 min. C. utilis was also found to absorb various L-amino acids rapidly without producing any significant decrease in the half-life of glutathione. While the gamma-glutamyl cycle is thus operating in C. utilis, the rate of degradation of glutathione is found to be 100 times too slow for the cycle to be mediating the transport of these amino acids.     

枯草芽孢杆菌内切木聚糖酶的分离纯化与鉴定

目的：建立一种简便、快速的木聚糖酶分离和提取方法。方法：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,分离纯化枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中的木聚糖酶,进一步用薄层色谱和高压液相色谱对木聚糖酶进行鉴定。结果：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,从枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中分离得到了两种内切木聚糖酶,酶解桦木木聚糖的产要产物以木二糖和木三糖为主。结论：活性聚丙烯酰胺凝胶电泳和均质提取法相结合是一种新的分离纯化木聚糖酶的简便、有效方法。     

Enzymes metabolizing dimethylamine, trimethylamine and trimethylamine N-oxide in the yeast Sporopachydermia cereana grown on amines as sole nitrogen source

Abstract Sporopachydermia cereana , an ascosporogenous yeast, grew on dimethylamine, trimethylamine or trimethylamine N -oxide as sole nitrogen sources and produced mono-oxygenases for dimethylamine and trimethylamine that were significantly more stable than the corresponding enzymes found in Candida utilis . No trimethylamine mono-oxygenase activity was found in S. cereana grown on dimethylamine. In cells grown on trimethylamine N -oxide (but not on the other nitrogen sources), evidence for an enzyme metabolizing the N -oxide, possibly an aldolase, but more probably a reductase was obtained. All these activities showed a similar requirement for the presence of FAD or FMN in the extract buffer during isolation to retain activity. Amine mono-oxygenase activities showed a similar sensitivity to inhibitors, including proadifen hydrochloride and carbon monoxide as the corresponding enzymes in C. utilis . The trimethylamine N -oxide-dependent oxidation of NADH was more sensitive to inhibition by EDTA, N -ethylmaleimide and β-phenylethylamine than the mono-oxygenases, and less sensitive to KCN, and activity was significantly higher with NADPH than was observed with the 2 mono-oxygenases.