JPS626678A - Production of thermostable glucoamylase - Google Patents

Abstract

PURPOSE:To obtain the titled amylase useful as an sacchrifying enzyme for starch hydrolysis or alcohol fermentation industry, having a specific fittest reaction temperature, by cultivating a thermostable mold belonging to the genus Aspergillus, capable of producing thermosatable glucoamylase. CONSTITUTION:A variant of Aspergillus awamori is inoculated into a nutritive medium obtained by blending grain four such as wheat or its starch, potato starch or grain waste such as white rice bran, etc., with an organic or inorganic nitrogen source such as peptone, nitrate, etc., and an inorganic salt such as KH2PO4, etc., and subjected to liquid culture by aerated stirring or solid culture at 20-45 deg.C for 2-7 days. Then, the culture mixture is treated by combination of filtration, extraction, centrifugation, alcohol precipitation method, chromatography, gel filtration, etc., to collect thermostable glucoamylase having about 4.2 fittest reaction pH, 2-8 stable pH range and 65-75 deg.C fittest reaction temperature.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing thermostable glucoamylase.

Glucoamylase (EC3, 2, 1, 3) is also called amyloglucosigase and is used to synthesize amylose, amylopectin,
It is known as an enzyme that acts on polysaccharides with α-1°4 glucosidic bonds, such as glycogen and dextrin, and cleaves them into glucose units from their non-reducing ends, and is used in the starch hydrolysis industry, alcohol fermentation industry, and alcoholic beverage production. It is an enzyme that plays an important role in the manufacturing industry.

Conventionally, microorganisms have been used as enzyme sources for stable and large-scale collection of glucoamylase, and in particular, Rhizopus delelner (R, delelner), Rhizopus niveus (R, n1veus), Rhizopus javanicus (R, javanicus), Aspergillus ni〃-(^, niger) and Aspergillus awamori (A.

Filamentous fungi such as C. awamori) and strains belonging to the genus Endomycopsis, which are yeast or yeast-like bacteria, are known as strong glucoamylase-producing bacteria.

However, the optimal reaction temperature of these produced glucoamylases is 55°C to 60°C, and no glucoamylase having an optimal reaction temperature of 60°C or higher has been reported.

In the starch hydrolysis industry, the saccharification process requires a long time of 48 to 72 hours at a temperature of more than 55°C to less than 60°C.

Furthermore, there is a risk of microbial contamination at temperatures below 55℃, and temperatures below 60℃
Above these conditions, enzyme deactivation occurs, so strict temperature control is required.

Therefore, the present inventors believed that if glucoamylase with an optimal reaction temperature of 60°C or higher could be obtained, it would have extremely economical effects such as shortening the saccharification reaction time and preventing microbial contamination, and the results of intensive research were obtained. As a result, the glucoamylase produced by Aspergillus kawachi (A, Kaiuacbi) is thermostable, and the optimum reaction temperature is above 65°C.
It was found that the temperature was less than 75°C, as shown in FIG. )
, arrived at the present invention.

This strain is Aspergillus awamori (8, au + a-
mori) and is considered to be a mutant strain of (Rikou, 28.10
6 (1950)), and is a well-known strain that is widely sold as the m strain of seed koji for shochu. Aspergillus awamori, which is said to be the parent strain, is known as a filamentous fungus that produces glucoamylase as mentioned above, but the optimal reaction temperature for glucoamylase is above 55°C and below 60°C (Agric, Biol, Chem. +41,2
149 (1977)), and no heat resistance was observed. The heat resistance of glucoamylase produced by this strain has not been reported at all, and this is the first finding discovered by the present inventors. Glucoamylase collected from the culture by culturing this strain has maximum activity at temperatures above 65°C and below 75°C. It can be expected that its advantages as a saccharifying enzyme will be demonstrated and the economic effect will increase.

When culturing this strain to produce glucoamylase,
The nutrient media used are corn, barley, wheat,
Grain flours such as rice or their starches, potato starches such as potatoes and sweet potatoes, and grain wastes such as wheat bran, white bran, and rice bran obtained when processing grains are used in appropriate combinations.

Furthermore, peptone, yeast extract, ammonium salts, nitrates, other organic or inorganic nitrogen sources and dipotassium phosphate,
Inorganic salts such as phosphoric acid-potassium and magnesium sulfate may be added as needed.

As a culture method, liquid culture using aeration agitation or shaking culture, or solid culture using wheat bran or the like can be performed. Cultivation may be carried out for about 2 to 7 days at a culture temperature in the range of more than 20"C to less than 45C.

Although this strain produces a large amount of citric acid during cultivation, the glucoamylase produced is acid-resistant (as shown in Figure 3), so neutralization during cultivation is not necessary. In order to collect glucoamylase-containing liquid from the culture after completion of culture, in the case of liquid culture, it is performed by means such as filtration and centrifugation. In the case of solid culture, extraction is performed with water or various salt solutions. This culture filtrate or extract is used as glucoamylase crude enzyme stock solution, and general enzyme fractionation methods such as alcohol precipitation method, ammonium sulfate precipitation method, chromatography using ion exchange resin, ion exchange cellulose, etc., Dell filtration method, etc. By combining these, glucoamylase can be collected. This strain produces two glucoamylases, glucoamylase I and glucoamylase H, both of which are thermostable and are included in the present invention.

Next, a glucoamylase extract was obtained by solid-state culturing Aspergillus kawachi using crushed and polished rice as a raw material, followed by concentration using an ultrafiltration membrane, DE 8-to-Biogel (Bio-Rad) chromatography, and Sephacryl S- 30
The enzymatic properties of partially purified glucoamylase I and glucoamylase II obtained by gel chromatography are described below.

(1) Crop It acts on various starches in grains and potatoes, and produces glucose well, but does not produce transfer sugars such as isomaltose, isomaltose, and liose.

Glucoamylase ■ has a greater effect on raw starch than glucoamylase I.

(2) Substrate specificity Acts on polysaccharides having α-1,4 glucosidic bonds, such as amylose, amylopectin, glycogen, and dextrin.

(3) Optimum reaction pH Add 0.51 i of 0.1 M Macllvain buffer and 0.11 l of enzyme solution to 1 pg of 2% soluble starch, and react at 40°C, 55°C, and 70″C. When the optimum reaction pH of this enzyme was determined, both glucoamylase I and ① were found to be around pl+4.2, and the lower the reaction temperature, the wider the range (Fig. 1, 2).
As shown in the figure. ) (4) Stable pH range The stable pH range of this enzyme was measured using pinequilpine buffer. As a result, both glucoamylase I and ① were stable over the entire measured pH range when treated at 30°C for 60 minutes. (See Figure 3.) (5) Optimum reaction temperature 2% soluble starch IRl and 0.2M acetate buffer (p
H4) 0.2if and enzyme solution 0. In addition to IRl, the amount of glucose produced at each temperature was measured. As a result, both glucoamylase I and glucoamylase II have an optimal reaction temperature around 70°C. (See Figure 4.) (6) Add 0.1z1 of temperature-stable enzyme solution to 0.28 acetate buffer (ptl 4
) was added to the mixture, left at each temperature for 10 minutes, and then immediately cooled, 0.1 piece of debris was removed, and the remaining activity was measured by a conventional method.

As a result, both glucoamylase I and ■ had a residual rate of 100% at 50°C, and a residual rate of about 71% at 65°C. (See Figure 5.) (7) Effects of inhibitors, etc. Table 1 shows the effects of various metal ions (2X10-1) on this enzyme.

Table 2 shows the effects of various inhibitors on this enzyme.

As is clear from Tables 1 and 2, both glucoamylase I and ① are slightly inhibited by Cu', Fe+, and Hg2+ ions, and are slightly activated by Mn'' ions. CO: ion was also slightly activated. Regarding the effects of various inhibitors, almost no effect was observed on glucoamylase I and ①.

Table 1 Effect of heavy metals (relative activity) CaCI298 94 CoS0. 113 99CuSo
, 72 72FeCl=
76 66FeSo,
97 94IgC1□ 83
56MgC1210299 HnC1□127 124 Pb (CI, C00) 2106 93ZnS
04100 97 Table 2 Effect of inhibitors (relative activity) Monoiodoacetic acid 2 97
106P-mercuric chlorobenzoate 1
96 9? sodium lauryl sulfate
1 100 1077 Phenylmethylsulfonyl tsulfide 1. 9.5 102N-Ethylmaleimide 10 94 103 The present invention will be explained below in more detail with reference to Examples, but the present invention is not limited thereto.

The glucoamylase activity measurement method used in the present invention is as follows:
It is as follows.

A method for measuring the glucose produced by reacting an enzyme with 2% soluble starch as a substrate at 40°C in an acetate buffer (Iwano, Fumon, Fuyo 2 Japan Brewery Association Magazine, 71.383 (1976)) ), 6
IJ in 0 minutes! l? The activity of producing glucose was defined as one unit. In addition, raw starch decomposition ability is determined by reacting an enzyme with wheat starch as a substrate in an acetate buffer at 30°C, quantifying the glucose produced in the same way as glucoamylase activity, and calculating the activity of producing IJg of glucose in 60 minutes by 1 It was taken as a unit. Furthermore, the amount of glucose produced at 70°C was
The value divided by that at 0°C was defined as the thermostability value of glucoamylase.

Example 1 10g of crushed and polished rice was steamed, left to cool, and Aspergillus.
M. kawachi was inoculated and cultured at 40°C for 48 hours. Add 100 d of 0.5% saline solution to extract the produced glucoamylase, and measure the glucoamylase and raw starch decomposition power in the extract.1. Ta.

Glucoamylase yielded 234 units/g of koji, and raw starch decomposition power was 14.2 units/y of koji. The produced glucoamylase had a heat resistance value of 4.15.

Example 2 Add 10 zl of water to 2 g of wheat bran and white rice bran! addition 12
7 to the medium obtained by autoclaving at 0°C for 5 minutes.
Supergillus kawachi was inoculated and cultured at 40°C for 5 days. After completion of the culture, 0.5% saline 100111 was added to extract the produced glucoamylase. Glucoamylase in the extract was at position 574/S bran. The thermostability value of the produced glucose amylase was 3.34.

Example 3 Add 100 pieces of water to 8g of wheat bran and 2g of white rice bran, add 12
The medium obtained by autoclaving for 15 minutes at 0'C was inoculated with 7 Supergillus kawachi bacteria, and cultured with shaking at 40C for 5 days. After the culture was completed, it was filtered to obtain a culture filtrate.

Gluamylase in the culture solution was up to 219 units/life. The heat resistance value of the produced glue; amylase is 5.
It was 32.

Example 4 Using crushed and polished rice as a raw material, Aspergillus kawachi was inoculated and cultured at 32°C to 43°C for 2 days.

Add 2 kg of this rice malt to 0.5% saline (acetic acid buffer 100%
m M (pH 5, o)) 81 and the produced glucoamylase was extracted. Extract 7 by centrifugation and filtration
05611 was obtained. This extract contained 30.6 units of glucoamylase/z1. This extract was filtered using an ultrafiltration module (molecular weight cutoff 10) manufactured by Asahi Kasei Industries, Ltd.
,000) and ultrafiltration membrane YM of Amifun Co., Ltd.
-10 (molecular weight cut off 10,000) was used for ultraconcentration to 40 al. When this concentrated solution was subjected to ion exchange chromatography using DEAE-BioDel A (Bio-Rad), glu; amylase was separated into I and ■. Glucoamylase I was obtained at 420x (l) and contained 149 units/shoulder β. The specific activity increased approximately 50 times with 40% of the total activity, and α-amylase activity, transglucosidase activity, acid protease activity and Almost no acidic carboxypeptidase activity was observed. In addition, 735zf of gluamylase was obtained, containing 128 units of 7xi (60% of the total activity, but α-amylase was mixed in). Therefore, gel chromatography was further performed using Sephacryl S-300 (7 Almacia) to separate a-amylase. Glucoamylase ■ was obtained with 161i+N,
It contained 9 units/I11.

The specific activity increased approximately 63 times, and α-amylase activity, transglucosidase activity, acid protease activity, and acid carboxypeptidase activity were hardly observed. The raw starch decomposition activities of glucoamylase I and H are 0.48 units/IIl and 2.80 units/7ml, respectively.
Met. The thermostability value of glucoamylase I was 3.8, and the thermostability value of glucoamylase II was 5.2.

[Brief explanation of the drawing]

Figure 1 shows the optimum reaction pH of glucoamylase I and the
The figure shows the optimum reaction pH of glucoamylase ①. Figure 3 shows the 1)11 stability of glucoamylase I and H at 30°C.
, shows the results of investigation under 60 minute treatment conditions. Figure 4 shows the optimal reaction temperatures for amylase I and H.
, The results of the investigation under the conditions of O are shown. FIG. 5 shows the thermostability of glucoamylase I and ■ at pl+4.0.
The results of investigation under 10 minute processing conditions are shown. Gunshi] Agura; ゛ 工 ey) "n" 10,000 ≧ L 15 PF'l K 711 paper 3 figure H gun ra: 17e ra'tZ:' p) 4% ray @ 11
C300C, GO4) Figure 4 Hot spring (0C)

Claims (1)

[Claims] A method for producing thermostable glucoamylase, which comprises culturing a thermostable glucoamylase-producing bacterium belonging to the genus Aspergillus to produce thermostable glucoamylase whose optimal reaction temperature is 65°C to 75°C, and collecting the same.