U.S. patent number 4,559,307 [Application Number 06/542,846] was granted by the patent office on 1985-12-17 for treatment of yeast cells with proteolytic enzymes.
This patent grant is currently assigned to Phillips Petroleum Company. Invention is credited to Thomas R. Hopkins.
United States Patent |
4,559,307 |
Hopkins |
December 17, 1985 |
Treatment of yeast cells with proteolytic enzymes
Abstract
Functional protein having reduced nucleic acid content is
produced without initial denaturation of the protein by contacting
undenatured yeast cells with an alkaline protease at a temperature
of about 20.degree. C. to 40.degree. C. for about 2 minutes to 2
hours at a pH of about 8 to 11. The yeast cells are preferably
Pichia pastoris and the alkaline protease is preferably from
Bacillus lichenformis.
Inventors: |
Hopkins; Thomas R.
(Bartlesville, OK) |
Assignee: |
Phillips Petroleum Company
(Bartlesville, OK)
|
Family
ID: |
24165524 |
Appl.
No.: |
06/542,846 |
Filed: |
October 17, 1983 |
Current U.S.
Class: |
435/272; 426/60;
435/222; 435/267; 435/68.1; 435/938 |
Current CPC
Class: |
C12N
1/005 (20130101); C12N 1/08 (20130101); Y10S
435/938 (20130101) |
Current International
Class: |
C12N
1/00 (20060101); C12N 1/08 (20060101); C07K
003/12 (); A23L 001/28 (); C12N 001/16 () |
Field of
Search: |
;426/60
;435/69,222,255,256,267,272,938 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Viikari et al., "Reduction of Nucleic Acid Content of SCP", Process
Biochemistry, May 1977, pp. 17-19 and 35..
|
Primary Examiner: Naff; David M.
Claims
I claim:
1. A process for the recovery of functional protein from
undenatured yeast cells which comprises contacting said undenatured
yeast cells with an alkaline protease at a temperature of from
about 20.degree. C. to about 40.degree. C. for about 2 minutes to 2
hours and at a pH of from about 8 to about 11.
2. A process according to claim 1 wherein said yeast cells are from
the genera Pichia.
3. A process according to claim 2 wherein said yeast cells are
Pichia pastoris.
4. A process according to claim 1 wherein said alkaline protease is
derived from the genera Bacillus.
5. A process according to claim 4 wherein said alkaline protease is
derived from Bacillus lichenformis.
6. A process according to claim 1 carried out at a temperature of
from about 35.degree. C. to about 40.degree. C.
7. A process according to claim 1 carried out at a pH of 9.
Description
This invention relates to the treatment of microbial cells with
alkaline proteolytic enzymes to form soluble protein material
having good functional properties and a low nucleic acid
content.
The desirability of protein having good functional properties is
known in the art. Good functional properties for proteins are
important because they relate to the flavor, color, solubility,
thermal stability, emulsifying, foaming, and texturizing
characteristics of the protein. It is generally recognized that
protein fractions which are soluble have better functional
properties than insoluble fractions.
Furthermore, it is known that microbial cells or single cell
protein contain certain quantities of nucleic acids. The propriety
of using these microbial cells for food and feed purposes is
limited by the presence of nucleic acids which may cause certain
pathologic effects such as arthritis and urinary calculus.
The prior art such as U.S. Pat. No. 4,107,334 shows that previously
heat-denatured protein may be subjected to enzyme/heat treatment in
order to improve the solubility and hence functional properties of
the protein. Denaturation of the protein, of course, also requires
heat treatment to temperatures as typically high as 150.degree. C.
which is very energy intensive and may cause undesirable
degradation or cross-linking of the protein. It thus would be
highly desirable if in some instances it was possible to do away
with the denaturation step and yet achieve some solubilization of
the protein as well as a reduction of the nucleic acid content
therein.
It is therefore an object of the present invention to provide a
simple and improved process for preparing functional protein
reduced in nucleic acid content.
Other aspects, objects, and advantages of the present invention are
apparent from the specification and claims.
In accordance with the present invention I have discovered that by
treating yeast cells with an alkaline protease at a temperature of
from about 20.degree. C. to about 40.degree. C. for about 2 minutes
to 2 hours that partial solubilization of protein content of the
cells occurs resulting in a protein fraction of reduced nucleic
acid content with good functional properties. This is accomplished
even though the previous denaturation step known in the prior art
has been completely eliminated.
In accordance with the present invention, yeast cells are
utilized.
Suitable species of yeasts include species from the genera Candida,
Hansenula, Neurospora, Rhodotorula, Torulopsis, Saccharomyces,
Schizosaccharomyces, Pichia, Debaryomyces, Kluyveromyces,
Lipomyces, Cryptococcus, nematospora, and Brettanomyces.
Examples of suitable species include:
Candida boidinii
Candida utilis
Candida robusta
Candida rugosa
Hansenula minuta
Hansenula californica
Hansenula silvicola
Hansenula wickerhamii
Hansenula glucozyma
Hansenula nonfermentans
Torulopsis candida
Torulopsis versatilis
Torulopsis molishiana
Torulopsis nitratophila
Pichia farinosa
Pichia membranaefaciens
Pichia pastoris
Candida mycoderma
Candida stellatoidea
Candida claussenii
Brettanomyces petrophilium
Hansenula saturnus
Hansenula mrakii
Hansenula polymorpha
Hansenula capsulata
Hansenula henricii
Hansenula philodendra
Torulopsis bolmii
Torulopsis glabrata
Torulopsis nemodendra
Torulopsis pinus
Pichia polymorpha
Pichia pinus
Pichia trehalophila
Rhodotorula rubra
Presently preferred yeast are those from the genus Pichia, with
Pichia farinosa, Pichia membranaefaciens and Pichia pastoris most
preferred.
The above described microorganisms can be grown in a batch or
continuous fermentation process in the presence of oxygen, a source
of carbon and energy, and an assimilable source of nitrogen.
Various types of fermentation processes known in the art can be
utilized. For example, a foam-filled fermenter such as described in
U.S. Pat. No. 3,982,998 can be used.
Oxygen can be supplied to the fermentation process in the form of
air or oxygen enriched air. The source of nitrogen for the
fermentation can be any organic or inorganic nitrogen-containing
compound which is capable of releasing nitrogen in a form suitable
for metabolic utilization by the growing organism. Suitable organic
nitrogen compounds include, for example, proteins, amino acids,
urea, and the like. Suitable inorganic nitrogen sources include
ammonia, ammonium hydroxide, ammonium nitrate, and the like.
The sources of carbon used with the above microorganisms can be any
carbohydrate or starch containing material. For example, glucose
(the hydrolysis product of starch), sucrose containing sugars, or
hydrolyzed sucrose can all be utilized in the present invention.
Straight chain alcohols having from 1 to 16 carbon atoms per
molecule are utilizable as a carbon feedstock. Preferably the
alcohol has from 1 to 6 carbon atoms per molecule and more
preferably the alcohol will be either ethanol or methanol and most
preferably, methanol. Examples of suitable alcohols include
methanol, ethanol, 1-propanol, 1-butanol, 1-octanol, 1-dodecanol,
1-hexadecanol, 2-propanol, 2-butanol, 2-hexanol, and the like.
Mixtures of alcohols can also be employed if desired. Other
oxygenated hydrocarbons such as ketones, aldehydes, acids, esters,
and ethers are also suitable substrates and these usually have from
1 to 20 carbon atoms per molecule. Normal paraffins having from 1
to about 20 carbon atoms per molecule can also be used as
substrates.
Sufficient water is maintained in the fermentation so as to provide
for the particular requirements of the microorganism employed.
Minerals, growth factors, vitamins, and the like generally are
added in amounts which vary according to the particular requirement
of the microorganism and are generally known to those skilled in
the art or are readily determined by those so skilled.
The growth of the microorganism is sensitive to the operating
temperature of the fermentor and each particular microorganism has
an optimum temperature for growth. Exemplary fermentation
temperatures are in the range of about 20.degree. C. to about
60.degree. C.
Fermentation pressures are generally within the range of about 0.1
to about 100 atmospheres (10.13 to 10,132 kPa), more usually about
1 to 30 atmospheres (101.3 to 3,039 kPa), and more preferably about
1 to about 5 atmospheres (101.3 to 506.5 kPa) since the higher
pressures mean a greater level of dissolved oxygen in the aqueous
medium and usually higher productivities.
Any alkaline protease known in the art is suitable in the present
invention. They may be of either microbial, plant, or animal
original. Preferred are alkaline proteases of bacterial origin such
as those derived from Bacillus species. Particularly suitable are
those alkaline proteases derived from B. licheniformis.
The particular alkaline protease used must be present in an amount
sufficient to solubilize the yeast cell single cell protein to the
maximum desirable yet feasible level. As concerns the level of
proteolytic activity used in the present invention, the maximum
level is determined in part by economics.
Generally the temperature range for microbial cell/enzyme contact
is from about 20.degree. C. to about 40.degree. C., preferably from
about 35.degree. C. to about 40.degree. C. The exact temperature
employed will vary with the specific type of alkaline protease
used.
Generally the reaction time is from about 2 min. to about 2 hours,
preferably from about 15 minutes to about 60 minutes.
The use of an alkaline protease indicates, of course, that the
reaction is carried out in a basic medium. Preferably, though, the
pH should be from about 8 to about 11 and most preferably about 9.
The exact pH will vary with the specific type of alkaline protease
and SCP substrate used in the present invention.
It is within the scope of the present invention to immobilize the
particular protease used on the SCP substrate as is known in the
art. In this manner, the enzymatic digestion of the SCP could be
carried out on a continuous basis in a countercurrent process or
immobilized bed.
The following examples illustrate the present invention.
EXAMPLE I
A series of experiments were conducted in order to determine the
effect that Alcalase enzyme has on single cell protein (SCP)
extracts obtained from Pichia pastoris. Alcalase is a microbial
alkaline protease derived from B. lichenformis supplied by Novo
Enzyme Corp., Mamaroneck, N.Y.
In each experiment, 10 ml of 10% Pichia pastoris yeast suspensions
(packed cell volume/volume) was prepared, the resulting suspension
was adjusted to pH 9 and 0.25 mL of Alcalase 0.6 L was added. The
mixture was incubated for one hour at the temperature indicated in
Table I below.
The resulting treated SCP samples were centrifuged to sediment the
remaining solids, carefully drained of the supernatant and
resuspended to their original volume in water. The resuspended
solids were analyzed for protein content, nucleic acid content and
dry measure (DM) weight.
The results of each experiment are listed below in Table I.
TABLE I
__________________________________________________________________________
In Solids Fraction After Digestion Calculated and Centrifugation mg
NA in 1 g Incubation Protein Nucleic Acid DM Solubilized Expt. SCP
Preparation Temp. (.degree.C.) Remaining, % Remaining, % Wt. (g/l)
Protein***
__________________________________________________________________________
1 (Control) Fresh cell cream, 4.degree. 100* 100** 66.6 0 no added
enzyme 2 (Control) Fresh cell cream, 40.degree. 82 100 60.8 0 no
added enzyme 3 (Control) Fresh cell cream, 65.degree. 74 71 55.8
120 no added enzyme 4 (Invention) Fresh cell cream + 40.degree. 76
98 56.3 10 Alcalase Enzyme 5 (Prior Art) Fresh cell cream,
40.degree. 35 91 38.4 15 heat denatured for 2 min. at 65.degree.
C., then cooled to 40.degree. C. and Alcalase added
__________________________________________________________________________
*Control (Expt. 1) was 40.8 mg/ml protein **Control (Expt. 1) was
4.5 mg/ml N.A. ***[4.5 (mg/ml)solids N.A. concentration
(mg/ml)]/[40.8 (mg/ml)solids Protein concentration (mg/ml)]-
The above data indicate that the inventive treatment (Exp. 4),
which does not involve protein denaturation before incubation
results, not only in partial solubilization of the protein content
but also results in lesser amounts of nucleic solubilized than in
experiments involving a previous protein denaturation step (Run 5).
Thus, the data show the benefits of eliminating the previous
protein denaturation step in the preparation of soluble, protein
digest.
EXAMPLE II
Effect of pH of Incubation
Samples of fresh cell cream were treated as in Example I,
experiment 4 except that the pH during proteolytic digestion was
adjusted to different values. All incubations were done at
40.degree. C. for a period of one hour.
The results are given below in Table II.
TABLE II ______________________________________ In Solids Fraction
After Digestion and Centrifugation* Protein Nucleic Acid Expt.
Incubation pH Remaining, % Remaining, %
______________________________________ 1 7 90 87 2 9 76 91
______________________________________ *Contents relative to
control (Example I, Expt. 1).
The above results show that protein digestion using alkaline
protease proceeds best at higher pH values.
Reasonable variations and modifications are possible in the scope
of the foregoing disclosure and the appended claims.
* * * * *