JPH0732701B2 - Salt-tolerant ethanol-producing yeast - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a yeast strain belonging to the genus Candida , which can produce ethanol with high efficiency even in a medium having a high salt concentration.

[0002]

2. Description of the Related Art In fermentative production of ethanol, inexpensive fermentation raw materials, improvement of fermentation yield, reduction of cooling water, reduction of distillation energy and the like are important in order to reduce the production cost. Particularly important factors in the fermentation stage include high-concentration charging and high-temperature fermentation.

Molasses which is preferably used for ethanol fermentation contains various salts such as potassium, magnesium and sodium, the amount of which varies depending on the origin of the raw material, but generally about 0.5-1.5M. Is the concentration of. When high-concentration fermented materials were charged, the salt concentration in the medium could be 1.0 M or higher, which could cause problems with yeast growth inhibition and fermentation inhibition. Therefore, it has been desired to obtain a salt-tolerant high ethanol-producing yeast.

On the other hand, Saccharomyces ( Saccharomyc) is exclusively used for ethanol fermentation using molasses as a raw material.
es ) yeast has been used, and breeding and improvement have been continued in order to obtain salt-tolerant yeast belonging to the genus Saccharomyces, but a sufficient salt-tolerant yeast strain has not yet been obtained. Moreover, Saccharomyces cerevisiae ( Saccharomyces cerevisiae) resistant to high salt concentration
There is also an example in which e ) was obtained by breeding and improvement (Kondo et al., Nagano Shokuko Shiho, Vol. 15, p. 95, (1987)), but it was not sufficient for industrial use.

[0005]

Therefore, an object of the present invention is to provide a yeast strain capable of highly efficiently producing ethanol even in a medium having a high salt concentration.

[0006]

Means for Solving the Problems In order to solve the above problems, the present invention separates various yeasts from soils in Japan and overseas, plants such as flowers, fermented foods and fermented beverages, As a result of diligent search for yeasts having salt tolerance and capable of carrying out ethanol fermentation with high efficiency, yeast Nos. Belonging to the genus Candida isolated from soil in Taiwan were identified. 3110 and yeast No. 2436 belonging to the genus Candida isolated from sugar cane of Okinawa were found to be strains meeting the above-mentioned problems, and the present invention was completed. [No. 311
Taxonomic property of strain 0] This strain is a strain isolated from soil collected in Taiwan, Taiwan on September 28, 1988, and has the following properties.

It has an egg-shaped or spherical shape of 3 to 7 μm and has no mycelium-like structure. No spores are formed in any of the gypsum, Klein medium and malt extract medium. Example 1
As shown in FIG. A. Barnett
Rayoru East: Characterization and Identification (chara)
categorization and identifying
cation), various physiological properties were examined. As a result of a comparative examination of this result with a known yeast strain,
There is no matching strain, Torulaspo
ra ) The possibility of a new type of yeast, which is a non-spore generation of yeast, was considered. As a result of analysis by pulse field chromosomal DNA electrophoresis to examine homology with the yeast of the same genus, five chromosomal DNAs of 1000 kb or more were confirmed, and T. Del Bruecki ( T. delbruecki
i ), T. The pattern was similar to that of globusa ( T. globosa ), and it was speculated that it is closely related in terms of classification (Example 2). However, as a result of the homology analysis by DNA hybridization, the homology with them was 40-.
It was about 50% (Example 3), where this strain was classified as Candida (Candida) spp yeast, Candida Dafa Men Ti Lee (Candida ferme
ntii ).

[No. Characteristics of 3110 strain] As described in Example 4, salt resistance as seen with NaCl, KCl, glucose resistance as seen with glucose, organic acid resistance as seen with sodium propionate, and antibiotic resistance such as cycloheximide, amphotericin B, hygromycin,
S. cerevisiae used as a control strain Cerevisiae
iae ) IFO 0224 strain was remarkable. 2. Those tolerances were also exhibited, for example, in an ethanol fermentation system in a liquid culture containing 1 M sodium chloride as described in Example 5. 3. As described in Example 6, the fermentation process was also excellent in the molasses medium. When molasses produced in Indonesia was diluted twice with tap water and cultured in a medium containing 0.2% ammonium sulfate, the result was superior to that of the control IFO 0224 strain.
When various sugars in the culture solution on the 7th day of culture were quantified by high performance liquid chromatography, as shown in Example 7, there were many sucrose residues, while other sugars such as IFO 0224 and other sugars served as controls. It was considered that the invertase activity was less than that of 1953 and was at the regulation stage of the fermentation rate.

[No. Taxonomical properties and characteristics of strain 2436] This strain is a strain isolated from sugar cane collected in Okinawa Prefecture. When the 2436 strain was examined for various physiological properties such as assimilation and fermentability tests, analysis of chromosomal DNA by pulse field electrophoresis, various resistance tests, and culture tests, the size of chromosomal DNA was small. Although there is a difference, the property is extremely no. Three
It was similar to 110 and could be judged to be the same species.

The inventors of the present invention dated July 6, 1991, and selected No. 3110 to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, with the consignment number Micro Engineering Lab 12348 (FERM P
-12348).

[0011]

EXAMPLES Next, examples will be described. Example 1 J. A. Yeast: Characterization and Identification by Barnett et al.
ation and identification)
No. 2 according to the second edition of Taxonomic identification of the 3110 yeast strain was performed. The results of examination of various physiological properties are shown in Table 1. The number of side chains of ubiquinone is 6 units, and G + C of nuclear DNA
The content (mol%) was 44.9%.

Table 1. No. Physiological properties of 3110 strain 1) Fermentability test D-glucose + D-galactose-maltose-methyl α-D-glucoside-sucrose + α, α-trehalose-melibiose-lactose-cellobiose-meletitose-raffinose + inulin + Starch-D-xylose-2) Growth test D-glucose + D-galactose-L-sorbose + D-glucosamine-D-ribose-D-xylose-L-arabinose-D-arabinose-L-rhamnose-sucrose + Maltose-α, α-trehalose + methyl α-D-glucoside-cellobiose-salicin-arbutin-melibiose-lactose-raffinose + meletitose-inulin + starch-glyceros + Erythritol-ribitol-xylitol + L-arabinitol-D-glucitol-D-mannitol + galactitol-myoinositol-D-glucono 1,5 lactone + 2-keto-D gluconic acid + 5-keto-D gluconic acid-D -Gluconic acid-D-glucuronic acid + D-galacturonic acid-DL-lactic acid + succinic acid-citric acid-methanol-ethanol-propane 1,2 diol-butane 1,2 diol-potassium nitrate-potassium nitrite-ethylamine-L-lysine + Gadaverine D Creatine-Creatinine-Glucosamine + Vitamin deficiency + 3) Resistance test 0.01% Cycloheximide-0.1% Cycloheximide -1% Acetic acid -50% D-Glucose + 60% D-Glucose + 4) Growth temperature 25 ° C + 30 ° C + 35 ° C-37 ° C-40 ° C-4) Additional test DBB reaction-D; Growth is delayed.

Fermentation and proliferation test methods are described on page 5 of the same document.
I obeyed. Ubiquinone side chain number measurement is agricultural
And Biological Chemistry (AgriBio
l. Chem) Vol. 37, No. 3, 621, (197).
3 years) and confirmed by reverse phase thin layer chromatography
It was The GC content measurement is published by the Academic Publishing Center, Kazuo Komagata
Chapter, Chemical Classification Experimental Method for Microorganisms, pp. 227-242
Method, Shimadzu spectrophotometer UV-2100 and temperature control
40 minutes at 60-100 ° C using roller SRP-8
Staining in 1 × SSC buffer during a linear temperature gradient between
A of color DNA₂₆₀Track the change and from the measured Tm value
Calculated by the formula of GC (%) = (TM-69.3) /0.41
It was As a result of comparing these results with known yeast strains,
There is no strain that doesTorulaspor
a) Considering the possibility of a new type of yeast that is a non-spore generation of yeast
Was given.

Example 2 Pulse field chromosomal DNA electrophoresis was performed to examine the homology with Torula spora yeast (FIG. 1). As a result, 1550, 1450,
Five chromosome DNs of 1350, 1200, 1020 kb
A can be confirmed, and T. Delbrueckii (delbruec
kii ) and T. It was a pattern similar to globosa , and it was inferred that it was closely related to classification. Method using CHEF-DRII Bio-Rad, instruction manual, S. Gel-embedded DNA was prepared by standard procedures for cerevisiae, and the DNA was purified using ultra pure DNA grade agarose (Ultra pu).
re DNA grade agarose, bio-rad) 1.0% gel, 0.5 x TBE buffer at 14 ° C,
It was confirmed by electrophoresis at 180 V and pulse time of 100 seconds for 20 hours and 150 seconds for 10 hours.

Example 3 Results of homology analysis by DNA hybridization (Table 2), No. 3110 strain and Torulaspora ( To
The homology with R. spp. ) yeast was 40-50%.

Table 2. DNA homology (%) Probe D NA HB101 0224 0955 1160 3110 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━ Solid E. coli HB10l 100 14.6 13.4 12.7 2.1 (Escherichia coli) S. cerevisiae IFO0224 16.8 100 19.3 12.0 18.9 (Serbysie) DT. 18.1 (Globosa) A No.3110 19.6 14.5 54.0 35.3 100

The method is described in Journal of General and Applied Microbiology (J. Gen. A).
ppl. Microbiol. ), 34, 191-199, (1988), denatured 1 μg / 100 μl of DNA purified by CsCl ultracentrifugation, and then dispensed and immobilized on a microtiter plate for enzyme-linked immunosorbent assay (ELISA). . Also, 10 μg of the same DNA
Is dissolved in 0.33 mg / ml of photobiotin (vector) and biotinized by irradiation with a 500 W mercury lamp for 15 minutes.
-Free photobiotin was removed with butanol.
After denaturation, the mixture was sonicated, and 1 ml of the hybridization solution was added to 50 μl thereof, and 100 μl was added to the immobilized DNA that had been previously treated in the prehybridization solution at 37 ° C. for 10 minutes, and hybridized at 45 ° C. for 2 hours. Hybridization solution is 20 x SSC
(3M NaCl (Wako); 0.3M sodium citrate (Wako)) 1.0 ml, 100 × Denhard (Denha
rdt: 2% BSA (Wako); 2% PVP (Merck); 2% Ficol (Fico1) 1400 (Pharmacia)) 0.1 ml; denatured salmon DNA (10 mg /
ml, Wako) 0.1 ml; formamide (Merck) 5.0 m
l; distilled water 3.8 ml; SDS (Sodium dode
cyl sulfate, Wako) 0.3 g. Pre-hybridization solution is 20 ml of SSC 1.0 ml;
100 x Denhard 0.5 ml; salmon DNA 0.2
ml; formamide 5.0 ml; distilled water 3.3 ml. After washing 3 times with 1 × SSC, a streptavidin-peroxidase solution (500 ng streptavidin-peroxidase (Sigma) / ml PBS (NaC
l 137 mM; KCl 2.7 mM; Na ₂ HPO ₄ 8
mM; KH ₂ PO ₄ 1.5 mM); BSA 0.5%; Triton X-100 0.1%) 100 μl, and added at 37 ° C.
It was treated with PBS for 10 minutes and washed 3 times with PBS. Substrate solution (o
-Phenylenediamine 3.7 mM ;, citric acid 24.3 m
M; Na ₂ HPO ₄ 51.4 mM, pH 5.0) 100 μ
1 and added, and treated at room temperature for 30 minutes. Stop the reaction by adding 50 μl of 2N H ₂ SO ₄ and 492 with a plate reader.
The absorbance at nm was measured. Relative similarity is OD-OD
_blank / OD _max- determined as OD _blank . OD _max
Is the value in the well where the strongest color development occurs.

Example 4 As shown in Table 3, No. 3110 strain is MY medium (yes
ast extract (Difco) 0.3%; mal
textract (Difco) 0.3%; Polypeptone (Nippon Pharmaceutical Co., Ltd.) 0.5%; Glucose (Domestic Chemistry) 1.0
%, PH 5.5). NaCl Demi was salt tolerance, impaired glucose salt viewed glucose, resistance to organic acid viewed with sodium propionate, and cycloheximide, amphotericin B, and antibiotic resistance to hygromycin were used as the control strain S. Cerevisiae ( c
erevisiae ) IFO 0224.

Table 3. Resistance test in agar medium IFO 0224 3110 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ NaCl 2.5 M --- 2-- + 1.5-++ 1 + ++ 0.5 + ++ KCl 3 − − 2.5 − + 2 ± + 1.5 + ++ 1 ++ + + 0.5 0.5 ++ ++ Glucose 3.2 − − 2.4 − ++ + 1.6 ++ + + 0 .8 ++++ Sodium propionate 0.8 ± 0.8 ± ++ 0.4 ++ Cyclo 3.12 μg / ml−− Heximide 1.56− + 0.78 − ++ 0.39 − ++ 0.20+ ++ Amphote 50 − − Lysine B 25 − + 12.5 − ++ 6.25 − ++ +3.12 − ++ +1.56 + ++ Blank ++ + + + ━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━ − No growth is observed + Growth is recognized ++ Vigorous growth is recognized

Example 5 No. The above resistance of strain 3110 was also exhibited in an ethanol fermentation system in liquid culture as shown in FIG. 2, for example. 1
YM medium (yeast extra) supplemented with M sodium chloride
ct 0.3%; malt extract 0.3%; b
actopepton (Difco) 0.5%; sucrose (Wako) 25%; KH ₂ PO ₄ (Wako) 0.2%; M
gSO ₄ · 7H ₂ O (Wako) 0.1%, pH5.5) 150
Rotate 250 ml Erlenmeyer flask containing 6 ml
When cultured at 125 rpm for 7.7 m / m, ethanol production of 7.7 v / v% was observed, and the fermentation rate and rate were remarkably excellent as compared with the IFO 0224 strain.

Example 6 As shown in FIG. 3, an excellent fermentation process was shown in the molasses medium. When molasses produced in Indonesia was diluted 2-fold with tap water and cultured in a medium containing 0.2% ammonium sulfate under the same conditions as in Example 5, the result was superior to that of the control IFO 0224 strain.

Example 7 In the course of culture shown in FIG. 3, various sugars in the culture solution on the 7th day were quantified by high performance liquid chromatography.
As shown in Table 4, many sucrose remained, while other sugars were used as controls, such as IFO 0224 and IFO 0224.
It is less than 953, and it is considered that the invertase activity is in the regulation stage of fermentation rate.

Table 4. Concentration of various sugars (g / l) on the 7th day of molasses culture Medium component IFO 0224 IFO 1953 No. 3110 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━ Glucose 114.4 71.6 87.5 68.7 Fructose 104.1 109.2 111.6 53.6 Sucrose 225.8 39.2 142.2 119.3 Trehalose 0.05 0 .31 0.08 tr melibiose 0.04 0.09 0.08 0.03 raffinose tr 0.23 0.07 0.05 ━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━ tr: Almost not detected.

[Brief description of drawings]

FIG. 1 is a diagram showing a pattern of chromosomal DNA subjected to pulse field electrophoresis.

FIG. 2 is a diagram showing the course of alcohol fermentation in YM medium.

FIG. 3 is a diagram showing the course of alcoholic fermentation in a molasses medium.

Claims (2)

[Claims] 1. Candida fermentii ( Ca
A yeast strain belonging to Ndida fermentii ) and capable of highly efficiently producing ethanol in a medium containing a high concentration of salts. 2. A method for producing ethanol using the yeast strain according to claim 1.