JPH0357747B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing glucoamylase.
More specifically, the present invention relates to a method for producing glucoamylase using red yeast belonging to the genus Rhodosporidium that has the ability to produce glucoamylase. Glucoamylase (EC3.2.1.3), also called amyloglucidase, is a
It is known as an enzyme that acts on polysaccharides with 1,4 glucosidic bonds and cleaves them into glucose units from their non-reducing ends. Conventionally, strains belonging to the filamentous fungi genus Rhizopus and Aspergillus have been known as microorganisms that produce glucoamylase. .
javanicus), Aspergillus niger (A. niger)
and Aspergillus awamori (A.awamori)
are known as strains with high glucoamylase production ability. Furthermore, strains belonging to the genus Endomycopsis and Endomyces, which are yeast or yeast-like bacteria, are also known as strong glucoamylase-producing bacteria. The present inventors have attempted to screen a wide variety of yeast strains that have higher glucoamylase producing ability than those found in nature, with the aim of obtaining saccharifying enzymes for sake brewing. As a result of intensive research, they succeeded in discovering a strain of yeast with high glucoamylase-producing ability from the soil around the starch factory. As a result of examining the mycological properties of this strain, it was found that it belongs to the genus Rhodosporidium, which is a type of red yeast. So far, yeast or yeast-like bacteria that produce glucoamylase include strains belonging to the genus Endomycocypsis and Endomyces, as well as Satucharomyces cerevisiae (S.
cerevisiae); Journal of Bacteriol., Vol. 138, pp. 1022-1025 (1979), Lipomyces kononenkoan (K.
kononenkoan)；European Journal
of Applied Microbiological Biotechnology (Eur.J.Appl.Microbiol.
Biotechnol.) Vol. 11, pp. 241-250 (1979), S. diastaticus;
Journal of Instructional Brewery (J.Inst.Brew.), Vol. 87, pp. 244-247 (1981), etc., are known to have the ability to produce glucoamylase in strains belonging to the genus Rhodosporidium. There have been no reports of this strain, and I learned that the glucoamylase collected from the culture by culturing this strain in an appropriate nutrient medium can be expected to be used not only as a saccharifying enzyme for sake brewing but also as an enzyme for producing glucose. It is a completed invention. Next, we will discuss the mycological properties of this strain. a Growth status on each medium This strain grows well on wort agar medium or MY agar medium, and its cell morphology varies in size.
It is 2.5μ×5μ and has an oval shape. The mode of growth is multipolar budding, and pseudohyphae or mycelia are formed, even after 2 weeks of growth.
No ballistospores were observed.
Also, although it does not form fissiles on wort agar, MY agar, and oatmeal agar,
teliospore, clamp connection
(clamp connection) is formed on oatmeal agar medium. b Physiological properties Assimilation of nitrate (+) Requirement for vitamins (-) Production of starch-like substances (-) Degradability of arbutin (+) Production of carotenoids (+) The pigment produced is α-carotene. Urine decomposition (+) No fermentation of glucose or sucrose. c Assimilation of various sugars Glucose + galactose + L-sorbose + sucrose + maltose + cellobiose + trehalose + lactose - melibiose - ruffinose + melezitose + inulin + soluble starch + D-xylose + D-ribose + L-rhamnose - ethanol + Glycerol + (weak) Erythritol - Ribitol + (weak) Inositol - D-mannitol + Galactitol + α-Methyl-D- D-Glutitol + Glucoside + Salicin + DL lactic acid - Succinic acid + (Weak) Citric acid - Above results Based on the description in The Yeast 2nd edition (1971) pages 803-814, this strain was found to belong to the genus Rhodosporidium, especially because it produces carotenoid pigments and does not form extruded spores. Rhodosporidium sp.J−
It was named 5B. And this strain is FERM-P No.
6948 and has been deposited at the Institute of Microbiology, Agency of Industrial Science and Technology. When culturing this strain to produce glucoamylase, corn, corn,
Husk powder or starch of barley, wheat, rice, etc., potato starch such as potato, sweet potato, tapiao, etc.
Various saccharides such as dextrins, oligosaccharides, disaccharides, and monosaccharides, and grain wastes such as wheat, white sugar, and rice sugar obtained during processing of shells are used in appropriate combinations. Furthermore, peptone, kinako, soybean meal, cornstarch liquor, organic and inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, and ammonium nitrate, and inorganic salts such as dipotassium phosphate, agnesium sulfate, and monopotassium phosphate, and yeast extract. It is also good to add micronutrients such as malt extract as needed. As for the culture method,
Liquid culture using aeration agitation or shaking culture, or solid culture using wheat or the like can be carried out. When using liquid culture as the culture condition, the initial pH is 4.
It is sufficient to carry out aeration stirring or shaking culture for 2 to 7 days at 20 to 40℃ in the range of ~7. In the case of solid culture, wheat with a moisture content of 30 to 70% is used at 20 to 40℃ for 2 to 7 days.
It is sufficient to culture for several days. After completion of culture, glucoamylase-containing liquid is collected from the culture by means of filtration, centrifugation, etc. in the case of liquid culture. In the case of solid culture, extraction is performed with water or various salt solutions. The crude glucoamylase in the containing solution can be further purified by a suitable combination of conventional purification means, such as alcohol fractionation, salting out, basic anion exchange chromatography, etc. Next, Rhodosporidium sp.
The enzymatic chemical properties of partially purified glucoamylase obtained by culturing P No. 6948 to obtain a glucoamylase-containing solution, followed by ammonium sulfate salting out and DEAE-cellulose column chromatography treatment will be described. (1) Action: Acts on various starches of grains and potatoes, produces glucose well, and does not produce transfer sugars such as isomaltose and isomaltotriose. It also acts on raw starch (wheat starch, durum starch) to produce glucose. (2) Substrate specificity Acts on polysaccharides with α-1,4 glucosidic bonds such as amylose, amylopectin, glycogen, and dextrin. (3) Add 0.1 ml of enzyme solution and 0.5 ml to each buffer with optimal pH of 0.4 ml.
2% soluble starch was added and the reaction was carried out at 40°C for 10 minutes to determine the optimum pH of this enzyme, which was found to be around pH 4 to 6 (shown in Figure 1). In addition, pH2.2-8.0
Macllvain buffer was used for the pH range of 8.0 to 10, and KoIthoff's buffer was used for the pH range of 8.0 to 10. (4) Stable pH range The stable pH of this enzyme is determined by using potassium hydrochloric acid buffer for the range of pH 1 to 2, and using MacIlvain for the range of pH 2.2 to 8.0.
The pH range of 8.0 to 10 was measured using Kolthoff's buffer solution. As a result, this enzyme
It was found that in the pH range of 4 to 7, it is stable even when left at 30°C for 24 hours (as shown in Figure 2). (5) Optimal temperature 0.1M acetate buffer (pH 5.0) in 0.1ml of this enzyme solution
0.4 ml of this enzyme was added and reacted for 10 minutes at each temperature to determine the optimum temperature for the enzyme. As a result, the optimum temperature for this enzyme was around 50°C (shown in Figure 3). (6) Temperature stability 0.1M acetate buffer (pH5.0) in 0.1ml of this enzyme solution
Add 0.4ml of
The temperature stability of this enzyme was determined by measuring the residual activity, and the enzyme was found to be stable even when treated at 47°C for 10 minutes (as shown in Figure 4). (7) Effects of inhibitors, etc. Various metal ions (10 ^-3 M) on this enzyme
Table 1 shows the effects of

【table】

[Table] Table 2 shows the effects of various inhibitors (10 ^-3 M) on this enzyme.

[Table] As is clear from Tables 1 and 2, this enzyme is significantly inhibited by N-bromosuccinimide and KMnO ₄ .
Fe ³⁺ , Cu ²⁺ , and Hg ²⁺ , monoiodoacetic acid,
Although it was slightly inhibited by EDTA, it was hardly inhibited by other metal ions. Furthermore, this enzyme, which has the above properties, even in the state of crude enzyme obtained from culture, has a concentration of 5%.
As a result of using paper chromatography to examine the saccharides that decompose and produce over time when acting on soluble starch, the production of isomaltose, isomaltotriose, etc. was not observed, and the majority of the produced sugars were glucose, indicating that oligosaccharides were not produced. Since almost no sugar production is observed, this strain of Rhodosporidium sp.
5B FERM-P No. 6948 was found to be a strain with high glucoamylase production ability. Therefore, the glucoamylase produced using this strain is optimal for glucose production. Furthermore, since it has the action of decomposing raw starch, it can be expected to be used as a future energy saving measure. The invention will be explained below with reference to examples. The amylase activity measurement method used in the present invention is as follows. 40 glucoamylase in 1% starch solution
After reacting at ℃ for 10 minutes, the reducing power generated was determined by the Somogyi method, and the enzyme activity was determined to be 1.0 IU when the reducing power was equivalent to 1.8 mg of glucose. Example 1 Rhodosporidium sp.J-5B FERM-PNo.6948
were grown on MY agar slants at 37°C for 2 days,
Part 1 Place a platinum loopful in advance in a 500ml Erlenmeyer flask with 100ml of a medium consisting of 5% wheat and 0.5% rice sugar.
(pH 5.5) and sterilized in an autoclave at 110°C for 20 minutes, the resulting medium was inoculated and cultured for 4 days at 30°C with shaking at 160 revolutions per minute. The glucoamylase activity of the supernatant obtained by centrifuging the culture solution is
It was 158I.U. Example 2 50ml medium consisting of 10% corn flour
(pH 5.5) into a 500 ml Sakaguchi flask, and
℃, after sterilizing for 20 minutes, pre-
Rhodosporidium sp.J−5B FERM−PNo.6948
One platinum loop of the culture that had been grown on the MY agar slant medium for 2 days was inoculated and cultured at 30°C for 4 days in a shaking incubator at 120 revolutions per minute. After the culture was completed, the total culture solution was reduced to 50 ml, and the glucoamylase activity was measured after filtration and found to be 134 I.U/ml. Example 3 Rhodosporidium sp.J-5B according to Example 1
FERM-P No. 6948 was cultured, and then 41 g of ammonium sulfate was added to 80 ml of a glucoamylase-containing solution obtained by filtration of the culture solution. The resulting precipitate was collected, dissolved in a small amount of water, dialyzed, and then placed on a DEAE-cellulose column. Processed by chromatography,
The unadsorbed fraction was collected in the column to obtain 50 ml of a liquid containing glucoamylase activity. The partially purified glucoamylase activity thus obtained was 200 I.U/ml. Example 4 After adding 300 g of wheat, 100 g of potato starch, and 10 g of water to a 25-year fermenter,
Add 100 ml of a medium consisting of 1% potato starch and 0.5% yeast extract to each of three 500 ml Sakaguchi flasks in advance to a culture medium that has been sterilized by steaming for 40 minutes under pressure of Kg/cm ² and cooled to 30°C.
Rhodosporidium sp.J−5B FERM−PNo.6948
Cultured at 30°C for 2 days was inoculated and cultured with aeration at 30°C for 65 hours. The culture solution was filtered to obtain 8.5 glucoamylase-containing solution. The glucoamylase activity of this containing solution was 150 I·U/ml. Example 5 In a 100 ml Erlenmeyer flask, 2% soluble starch, 0.7% polypeptone, 0.1% KH ₂ PO ₄ , MgSO ₄ .
Dispense 30 ml of a medium consisting of 0.05% _7H2O , 0.01% yeast extract, and 0.5% rice sugar, sterilize it at 110℃ for 20 minutes, and then subplant Rhodosporidium in a slant in advance.
One platinum loopful of sp.J-5B FERM-P No. 6948 was inoculated and cultured with aeration at 30°C at 160 revolutions per minute.
It was cultured for 5 days. The culture solution was filtered to obtain 25 ml of a solution containing glucoamylase activity of 90 I.U/ml. Example 6 Add 400 ml of water to 400 g of wheat and mix, then put into 4 2-volume Erlenmeyer flasks and heat at 120℃ for 30 minutes.
After sterilization, 100ml of medium consisting of 1% soluble starch and 0.5% yeast extract was placed in a 500ml Sakaguchi flask in advance.
ml Rhodosporidium sp.J−5B FERM−P No.
One platinum loop of 6948 was inoculated and cultured with shaking for 2 days.
10 ml was added and cultured stationary at 30°C for 4 days. After the culture was completed, 400 ml of water was added to each Erlenmeyer flask, stirred well, and the culture was filtered. The filtrate was collected to obtain a total of 1500 ml of glucoamylase-containing liquid. This solution contained 190 I.U of glucoamylase per 1 g.

[Brief explanation of drawings]

Figures 1, 2, 3 and 4 illustrate the present invention.
The optimum pH curve, pH stability curve, optimum temperature curve, and temperature stability curve of glucoamylase obtained by culturing Rhodosporidium sp. J-5B FERM-P No. 6948 are shown, respectively.

Claims (1)

[Claims] 1. A method for producing glucoamylase, which comprises culturing yeast belonging to the genus Rhodosporidium having the ability to produce glucoamylase in a nutrient medium, and collecting glucoamylase from the culture.