Method for recovery of intracellular material by disruption of microbial cells with carbon dioxide under pressure

This invention is a method to rupture microbial cells in order to recover intracellular material in the cells comprising: PA0 a) treating the cells with carbon dioxide under pressure sufficient to enter the cells for time sufficient to allow enough carbon dioxide into the cells to effect later rupture, then PA0 b) suddenly releasing the applied fluid pressure on the cells so that the outer wall or membrane of the cells is ruptured by the expansion of carbon dioxide within the cell. Preferably the remaining intracellular material of the cells is separated and recovered. Also the treatment can be in conjunction with lytic enzyme to increase rupture rates. Preferably the enzyme remains active and protein in the cells retains its native state in the ruptured cell suspension. The preferred time for treating is from between about one hour and about fifteen hours. It is also preferred to treat initially at a pressure of from above about 500 psi gage to about 5000 psi and a temperature of about 10.degree. to about 85.degree. C. For improved rupture efficiency, the treating is repeated at least once.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method for recovery of intracellular material 
by disruption of microbial cells with carbon dioxide under pressure. 
2. Background Art 
Microbial cells have long been recognized as an important source of 
commercially useful biochemicals, antibiotics, food and enzymes. With 
increasing demand of microbial products in industry and medicine, 
considerable efforts have recently been committed to develop technologies 
for production of intracellular microbial materials from microorganisms 
and various genetically altered cultures. Release of cell contents is also 
vital to many investigations of bacterial metabolism. In any case, the 
cell disruption is a necessary operation for recovery or isolation of 
protein, intracellular enzymes and organelles. A variety of disruption 
techniques had been developed, and some are available commercially. 
Mechanical methods appear to be favored at the present time by their 
economic advantages, although several non-mechanical methods, particularly 
the enzymatic lysis, have attracted great attention. However, many of 
these methods often degrade or denature proteins in the process. 
Yeast is the most widely accepted traditional food item which has been 
exploited as a potential protein source; however, despite its nutritional 
value and abundant supply, the potential of yeast as a major food source 
has been hampered by various problems. Ideally, yeast cell biomass should 
be consumed directly as food or as food ingredients, but the thick cell 
wall reduces the digestibility and bio-availability of protein. The cell 
wall substances often cause allergic responses, diarrhea, and flatulence. 
Even after the cell wall is removed, some lipid components contribute to 
off-flavors by oxidation, and more importantly, the functional properties 
of proteins are impaired in the process. Finally, the protein from single 
cells contains a high level of nucleic acids, which can cause uricacidemia 
and gout. 
The nature of the cell walls and their removal have been discussed in the 
literature. Autolysis (particularly, thio-activated autolysis) and 
enzymatic lysis are among the conventional techniques in wide use for 
disintegration of microbial cells, other than yeast, to recover 
intracellular enzymes. Although most microbial cell walls can readily be 
ruptured, yeast cell walls are very difficult to disintegrate. Selection 
of yeast strains with weaker cell walls and alkaline treatment can 
facilitate yeast protein recovery. Other methods, such as homogenization, 
freeze-thawing, and pressurization have been used to rupture the yeast 
cells. 
Among these methods, pressurization may be the least expensive if the 
pressure can be kept at a reasonably low range. Prior investigation of gas 
pressure for rupturing cells, but not yeast cells, has found that carbon 
dioxide was effective at 500 psi. 
Supercritical fluid (SCF) has recently demonstrated great potential in its 
applications to food and pharmaceutical industries. The fluid possesses a 
combination of "gas-like" and "liquid-like" properties. It penetrates like 
a gas, and functions like a liquid. Such unique characteristics of SCF 
have found broad applications in diverse areas, particularly for 
extraction and separation of natural products. Various prior studies have 
extensively applied SCF as a medium, in place of conventional solvents, in 
enzyme reactions. 
SUMMARY OF THE INVENTION 
This invention is a method to rupture microbial cells in order to recover 
intracellular material in the cells comprising: 
a) treating the cells with carbon dioxide under pressure sufficient to 
enter the cells for time sufficient to allow enough carbon dioxide into 
the cells to effect later rupture, then 
b) suddenly releasing the applied fluid pressure on the cells so that the 
outer wall or membrane of the cells is ruptured by the expansion of carbon 
dioxide gas within the cell. Preferably the remaining intracellular 
material of the cells is separated and recovered. Also the treatment can 
be in conjunction with lytic enzyme to increase rupture rates. Preferably 
the enzyme remains active and protein in the cells retains its native 
state in the ruptured cell suspension. 
The preferred time for treating is from between about one hour and about 
twelve hours. It is also preferred to treat initially at a pressure of 
from above about 500 psi gage to about 5000 psi and a temperature of about 
10.degree. to about 85.degree. C. For improved rupture efficiency, the 
treating is repeated at least once. Also the carbon dioxide can contain an 
entrainer to enhance the rupture efficiency. The entrainer can be selected 
from a group consisting of chemicals such as ethanol, ethylene, toluene 
and mixtures thereof. It is preferred in this method to have lytic enzyme, 
which hydrolyzes cell wall material, present in the cells. Examples of 
lytic enzymes are .beta.-glucuronidase, lysozyme, or glucanases. The lytic 
enzyme and the entrainer can both be present in or with the carbon dioxide 
or like fluid. 
Finally it is also expected that carbon dioxide can be replaced by a fluid 
selected from the group consisting of ethylene, ethane, propylene, propane 
and mixtures thereof in the process of this invention. 
This invention extends the application of SCF to a new area in which the 
SCF is used for disruption of microbial cells to produce protein. The 
technique is beneficial to yeast cells in particular, because the SCF is 
capable of extracting off-flavors and, possibly, nucleic acids in the 
process of cell disintegration. 
Although other fluids can be used, carbon dioxide was chosen in this work 
as a primary SCF because it is non-toxic, non-flammable, inexpensive and 
physiologically safe. The critical temperature of carbon dioxide 
(31.1.degree. C.) is just above ambient, which minimizes the problems of 
thermal degradation (or denaturation) of delicate biological materials and 
natural products. In addition, its phase behavior and other thermophysical 
properties that are needed for process analysis and development are well 
studied. 
The method of this invention differs from prior methods, inter alia, by 
rupturing microbial cells by the sudden release, optionally repeatedly, of 
the applied fluid pressure. This causes the fluid (SCF) in the cells to 
"pop" the cell walls. Release of the process pressure involves a sudden 
release of the applied fluid pressure which follows penetration of SCF 
into cells. The expansion of SCF within the cells when it is released 
(de-pressured) forces the breakage of microorganisms. It is simple, 
inexpensive, and, more importantly, non-injurious to enzyme activities. 
The functional properties of proteins are all preserved. Disruption rates 
are sensitive to such process variables as temperature, pressure, and 
addition of entrainers to the fluid. Under optimum conditions, over 80% of 
cell walls can easily be ruptured within an hour for a variety of 
microbial cells. The disruption is, however, not as effective for some 
other cells. A typical example is yeast cells, which have been described 
as one of the most robust and rigid of all microbial cell walls. However, 
this invention does disrupt yeast cells.

DESCRIPTION OF PREFERRED EMBODIMENTS 
Example I 
Microorganism 
Cells of Saccharomyces cerevisiae were purified from baker's yeast (Red 
Star) on YMP agar (DIFCO Laboratories, Detroit, Mich.) plates containing 
yeast extract (0.3% w/v), malt extract (0.3% w/v), bacto-peptone (0.5% 
w/v) and glucose (2% w/v). The purified yeast was maintained on a YMP agar 
slant as a stock culture. Cells were first transferred from slants to a 
500 mL flask containing 200 mL liquid YMP medium which contains 10% (w/v) 
glucose. The cultures were incubated at 30.degree. C. in a shaker with 
agitation (200 rpm). Fresh cultures were prepared daily with the same 
procedure. After 18 hours of incubation, the yeast cells were harvested 
and washed with distilled water. After the liquid was decanted, the 
pellets were ready for subsequent experiments. 
Carbon dioxide was purchased from Matheson Gas Products with a minimum 
purity of 99.99%. 
Apparatus 
A static apparatus was used in this work which consisted of two major 
components: a piston injector and a pressure vessel. Carbon dioxide was 
supplied from the gas cylinder to a Ruska pump (Model 2200, Ruska 
Instrument Corp., Houston, Tex.), which compressed CO.sub.2 to a desired 
pressure prior to injection into the pressure vessel. The design of the 
vessel was similar to that of the Kuentzel closure reactor vessel (Series 
KC single ended unit) of Autoclave Engineers, Inc., (Erie, Pa.) with an 
internal volume of approximately 6.6 mL. Another pressure vessel of 50mL 
internal volume (Model 1019 HC, Parr Instrument Co., Moline, Ill.) was 
also used to investigate the effects of excessive amounts of CO.sub.2 on 
cell disintegration. The vessel was rated at a maximum pressure of 2,200 
psi at 40.degree. C. The vessels and all other parts exposed to high 
pressures were made of stainless steel 316. A thermostated water bath was 
used to maintain the pressure vessel at experimental temperature. A 
pressure gauge was installed in the CO.sub.2 inlet of the vessel to read 
the pressure. 
Cell Disruption 
To start an experiment, one gram of wet cells was placed in the pressure 
vessel, and 1 mL Tris-HCl buffer solution (pH 7.0 and 0.05M) was added. In 
some experiments, 10 mg of .beta.-glucuronidase (EC 3.2.1.31, from Helix 
pomatia supplied by Sigma Chemical Co., St. Louis, Mo.) or 1 mL toluene 
was also added to the suspension. The vessel was then enclosed and 
immersed in the thermostated water bath at the experimental temperature 
(25.degree., 35.degree. or 55.degree. C.). The yeast cells in the vessel 
were agitated with a magnetic stirrer. After the temperature was 
equilibrated and all tubing connections were secured, carbon dioxide was 
injected into the vessel at a fixed pressure (1,000, 3,000, or 5,000 
p.s.i.) via a preheated or cooling coil. The yeast cells were exposed to 
carbon dioxide for a designated length of time. Carbon dioxide was then 
rapidly released, and 4 mL of Tris-HCl was immediately added to the vessel 
after it was opened. The cell suspension was centrifuged and the 
supernatant assayed for total protein and enzyme activities. 
Enzyme Assay 
Activity of yeast alcohol dehydrogenase (EC 1.1.1.1) was determined by the 
method of Vallee and Hoch in Proceedings of Nat'l Academy of Sciences, 
41(6), p.327 (1955). Invertase (EC 3.2.1.26) was assayed by adding 0.1 mL 
of 0.125M sucrose to 0.9 mL supernatant. The pH of the supernatant was 
adjusted to 4.5 by adding 0.1N HCl. The reaction was carried out for 3 
minutes, and followed by heating the reacted solution at 100.degree. C. in 
a water bath for 5 minutes. Glucose formation in the solution was measured 
by a Beckman Glucose Analyzer. One unit of invertase is defined as the 
enzyme which hydrolyses 1 micromole of sucrose in 1 minute at 55.degree. 
C. and pH 4.5. The activity of glucose-6-phosphate dehydrogenase (EC 
1.1.1.49) was determined by the method of DeMoss, Methods in Enzymology 
Vol.1 p.328 (1955). The method for determination of fumarase (EC 4.2.1.2) 
activity was described by Racker in Biochimica Et Biophysica Acta, 
4,211(1950), except that Tris buffer (pH 7.0, 0.05M) replaced phosphate 
buffer. The concentration of protein was estimated by Lowry's method, J. 
Biol. Chem. 193, 265 (1965), with bovine serum albumin as a standard. 
Results and Discussion 
The experiments were performed in both regions of subcritical and 
supercritical temperatures of carbon dioxide over the pressure range of 
1000-5000 psi. Experimental results in two different regions of 
temperature are presented separately in the Figures, described below. 
Carbon Dioxide at Subcritical Temperatures 
Experimental results were obtained at 25.degree. C. FIG. 1 shows the 
release of the soluble protein and the activity of alcohol dehydrogenase 
(ADH) as a function of disruption time at the experimental pressure of 
1,000 psi. In 15 hours, one gram (wet weight) of yeast cells released 
approximately 33 mg of soluble protein, which contained 160 units of 
alcohol dehydrogenase. By prolonging the disruption time to 24 hours, 
neither an increase nor a decrease in the amount of protein release and 
enzyme activity of alcohol dehydrogenase was observed. At higher CO.sub.2 
pressures, the length of disruption time to release maximal quantity of 
protein was significantly reduced, as indicated in FIG. 2. The maximal 
amount of protein release at 3,000 and 5,000 psi of CO.sub.2 occurred at 
12 and 5 hours, respectively, while the activities of ADH were preserved 
at about 160 units per gram of yeast cells. 
In a preliminary experiment, the same baker's yeast cells were disrupted by 
grinding them at 25.degree. C. with abrasive glass. The whole process was 
completed within one hour with a maximal amount of 33 mg of protein 
released from one gram of wet yeast cells. Although the mechanical method 
could disrupt yeast cells more efficiently than high-pressure CO.sub.2, 
the activity of alcohol dehydrogenase diminished to 100 units per gram of 
yeast cells. The loss of enzyme activity was expected, since no reducing 
agent or protease inhibitors were added in the rupturing process. 
Intensive localized heating effects in the process of mechanical 
disrupution also often ensue with enzyme denaturation. Other disruption 
methods were also performed in this work for the yeast cells. FIG. 3 
compares the rates of protein release by autolysis at atmospheric pressure 
with toluene and by enzymatic lysis with .beta.-glucuronidase. Both 
methods released about the same amount of protein as fast as high pressure 
CO.sub.2, but a significant loss of alcohol dehydrogenase activity was 
found. Results from 1,000 psi CO.sub.2 with addition of 1 mL toluene or 
10 mg .beta.-glucuronidase (to 1 g of yeast cells) are also shown in the 
figure for comparison. The effects of adding toluene and 
.beta.-glucuronidase on the rates of disruption became even more apparent 
at higher pressures of CO.sub.2, as will be discussed later. The results 
demonstrate that the presence of CO.sub.2 in the disruption process 
preserves the activities of alcohol dehydrogenase. The mechanism of this 
enzyme stabilization is under investigation; however, there are several 
possible explanations: (1) CO.sub.2 may alter the hydrophobicity of the 
environment which inhibits protease. It was reported that during the lysis 
of Schizosaccharomyces pombe with .beta.-glucuronidase, addition of 
protease inhibitor prevented the loss of xylose isomerase; and (2) under 
high pressures, dissolved CO.sub.2 lowers the pH value to where protease 
cannot function. Regardless of these postulations, this disruption method 
with CO.sub.2 can be a valuable technique to produce enzymes from yeast, 
if other enzymes can also be preserved. 
In addition to alcohol dehydrogenase, three other enzymes were assayed 
after cells were disrupted by high pressure CO.sub.2 with 
.beta.-glucuronidase. These were invertase, glucose-6-phosphate 
dehydrogenase, and fumarase which are located in different compartments of 
the yeast cell. When the pressure of CO.sub.2 was at 5,000 psi in the 
presence of .beta.-glucuronidase, these enzymes were released to their 
maximal levels in 90 minutes. FIG. 4 shows the activity of each released 
enzyme after two-hour exposure under various pressures. In this Figure 
(and in all Figures) "ADH" means alcohol dehydrogenase. At zero pressure, 
which corresponds to enzymatic lysis with .beta.-glucuronidase, the 
activity of each enzyme is lower than that under high pressure CO.sub.2. 
Similar results were obtained when autolysis in toluene and mechanical 
grinding with abrasive glass were employed to disrupt yeast cells. In all 
cases, the activities of these enzymes were enhanced under high pressure 
CO.sub.2. 
To examine the effects of excessive amounts of CO.sub.2 on the disruption, 
experiments were repeated at same conditions of pressure and temperature 
with a 50 mL vessel. No significant differences were found if the cell 
suspension was stirred in the course of disruption. 
Carbon Dioxide at Supercritical Temperatures 
Two temperatures (35.degree. and 55.degree. C.) were studied in the 
supercritical region of CO.sub.2. FIG. 5 compares experimental results of 
total protein release at 35.degree. C. with those at 25.degree. C. The 
temperature effects are not evident under CO.sub.2 pressure of 1,000 
p.s.i. However, protein release appears to approach its maximal amount at 
a significantly faster rate with supercritical CO.sub.2 at higher 
pressures. The activities of released enzymes were again preserved in the 
presence of CO.sub.2, without addition of reducing agents and protease 
inhibitors which are commonly used in the disruption process. The 
discussion presented earlier with subcritical CO.sub.2 is applicable to 
this temperature and, therefore, need not be elaborated. 
The enzyme activities began to decay at higher temperatures (&gt;35.degree. 
C.) and were almost completely lost at temperatures of above 55.degree. 
C., although the rates of protein release were sequentially improved with 
increments in temperature. 
Using high pressure CO.sub.2 to lyse microorganisms for recovery of 
intracellular protein in combination with cell lytic enzymes is a part of 
this invention. The phenomenon that pressurized CO.sub.2 can prevent the 
deactivation of the released enzymes in the presence of crude preparations 
of cell lytic enzymes may reduce the cost of protein isolation from single 
cells. Furthermore, it was observed that CO.sub.2 may have extracted the 
off-flavors from the ruptured cell suspensions, and the off-flavor 
compounds were removed when CO.sub.2 were released. 
EXAMPLE 2 
This work demonstrates a method to improve the disruption rates of cells by 
repeatedly releasing the applied fluid pressure within the cells in the 
midst of a disruption process. The effectiveness of this technique is 
illustrated by the experimental results of rupturing yeast cells, which 
have been described as having one of the most robust and rigid of all 
microbial cell walls. The same technique can be analogously applied to 
other microbial cells. 
Experimental 
Cells of Saccharomyces cerevisiae used in this work were purified bakers' 
yeast from Red Star. The preparation of yeast samples was detailed in 
Example 1. The yeast pellets of this work contain 10% less moisture than 
those used in Example 1. Carbon dioxide was purchased from Matheson Gas 
Products with a minimum purity of 99.99%. 
The apparatus of Example 1 was used, namely, a static treatment that 
consists of a piston injector to feed CO.sub.2 at the experimental 
pressure (1,000 or 3,000 psi) into a pressure vessel containing one gram 
of wet yeast cells. The vessel was immersed in a thermostated water bath 
to maintain at a constant temperature (25.degree. or 35.degree. C.). After 
yeast cells in the vessel were exposed to CO.sub.2 for a designated length 
of time, CO.sub.2 was released and the yeast cells were collected for 
subsequent assays of total protein and enzyme activities, as described in 
Example 1. In the experiments of repeated release of applied CO.sub.2 
pressure in the midst of an experimental run, CO.sub.2 was recharged into 
the vessel at the experimental pressure immediately after it was released. 
The operation was repeated more than once in some experiments with a time 
interval that was divided evenly over the duration of a complete run. 
Results and Discussion 
Experimental results were obtained at both regions of supercritical and 
subcritical temperatures of CO.sub.2 for comparison. FIG. 6 shows the 
release of soluble proteins as a function of exposure time to CO.sub.2 at 
35.degree. C., while FIG. 7 presents similar results at 25.degree. C. Both 
are under pressure of 1,000 psi. R(2) and R(1) in the figures denote that 
carbon dioxide was released and repressurized twice and once, 
respectively, in the midst of a complete disruption process. R(0) serves 
as a control operation. The improvement in disruption rates with repeated 
release of applied CO.sub.2 pressure is evident. Similar results were 
observed at 3,000 psi, as shown in FIGS. 8 and 9. FIG. 8 is at 35.degree., 
FIG. 9 at 25.degree. C. 
The disruption rates are sensitive to process temperature and pressure. An 
increase in temperature and/or pressure will facilitate penetration of 
CO.sub.2 into cells. Higher temperatures appear to enhance the transfer 
rate of CO.sub.2, and also relax the cell walls to ease the penetration. 
Cell breakage comes as a result of gas expansion within the microbial 
cells when the vessel pressure is suddenly released. The action is 
strengthened under higher pressures. Another effective means to reduce the 
resistance of microorganisms to disruption is by addition of cell lytic 
enzymes as entrainers to CO.sub.2 fluid. The operation of repeated release 
of fluid pressure can be used in combination with lytic enzymes to further 
improve the efficiency of cell disruption without deactivation of enzymes. 
Activities of various enzymes (alcohol dehydrogenase, invertase, 
glucose-6-phosphate dehydrogenase, and fumarase) in the ruptured cell 
suspension were assayed to ensure that the process preserved the 
functional properties of proteins in the presence of CO.sub.2. The results 
are very similar to those presented in Example 1. 
While the invention has been described in connection with what is presently 
considered to be the most practical and preferred embodiments, the 
invention is not limited to the disclosed embodiments but, on the 
contrary, is intended to cover various modifications and equivalents 
included within the spirit and scope of the following claims.