CS204148B1 - Method of producing enzyme of alpha-amylase - Google Patents

Description

The present invention relates to a process for the production of the .alpha.-amylase enzyme by the yeast Cryptococcus luteolus.

Currently, the enzyme alpha-amylase is obtained by isolation from the production medium of Bacillus substilis and Streptococcus aureofaciens. Alpha-amylase production is described in some strains of the former genus Endomycopsis. It is Endomycopsis capsularis (today Saccharomycopsis capsularis), described by H. Ebert (Folia microbiologica 11: 422—428, 1966], hereinafter) Endomycopsis fibuliger (today Saccharomycopsis fihuliger), Endomycopsis javanensis (today Arthroascus javanensis) is Endner synonym for Saccharomycopsis fibuliger) and Endomycopsis hordei (now Saccharomycopsis fibuliger var, hordei), described by E. Matsutani, M. Sato and F. Hatori (US Pat.

297 547) and finally in Schwanniomyces occidentalis and Schwanniomyces alluvius. Disadvantages of α-amylase production methods in these strains are the relatively low α-amylase activity, high concentration of carbon sources, and difficult biomass separation.

These disadvantages are eliminated by the method of α-amylase production on a liquid nutrient medium containing assimilable carbon sources, complex nitrogen sources and mineral nutrient salts, which is based on the fact that the Cryptococcus luteolus CCY 17-6-2b strain is cultivated on a nutrient-containing soil 0.8 to 3 wt. % corn starch at 20 to 38 ° C, for 48 to 120 hours, at pH 4.7 to 6.8, wherein the α-amylase enzyme is obtained by evaporating the growth pod, or by precipitation with ammonium sulphate, or by affinity purification on a cross-linked starch .

Said strain of Cryptococcus luteolus 17-6-2b is deposited in U.S. Pat. collection of yeasts and yeast organisms in Bratislava.

The original strain of Cryptococcus luteolus CCY 17-6-2, which is of saprophytic origin, was isolated from the genus. It was sieved three times by passage on a broth prepared from 8% epichlorohydrin-crosslinked amylose gel immersed in synthetic Yeast Nitrogen Base (0.6 g / 100 ml of pod at 20-38 ° C for 48-120 hours and pH 4.7-7). 6.8 The new strain was designated as Oryptococcufe luteolus CCY 17 ^ 6-2b.

The strain Cryptococcus luteolus CCY 17-6-2b has cells oval to elongated, little uniform. In liquid medium, the ring forms a slimy coating, on the solid environment it is slimy and the cream is yellowish. It does not ferment sugars. In addition to D-glucose, it assimilates D-galactose, maltose, sucrose, lactose, melibiosis, cellobiose, trehalose, melezitose, D-xylose, D-mannitol, L-lysine, raffinose. The polysaccharide, γ204148, formed around the cells gives a iodine-like reaction. It does not assimilate either 1-sorbose or potassium nitrate. To grow it needs vitamins in the environment. The optimum growth temperature is 23 ° C at 5 ° C or does not grow at 42 ° C. It produces urease, catalase activity is high, fluidizes gelatin and rapidly autolyses.

Advantages of the method of α-amylase production according to the invention are: relatively high α-amylase activity even when working with simple fermentation broths, with lower carbon source concentration than with fermentation broths, for example for Bacillus substilis; easier separation of biomass after the fermentation process; more advantageous character of the product of the amylolytic reaction catalyzed by the enzyme Cryptococcus luteolus (D-glucose content up to 34%) than that of Bacillus substilis α-amylase; possibility of using biomass for feed purposes.

Example 1

From the washed gel prepared from sintered α (1 - * 4) glucan according to AO No. 186 059, triangular profiles of 1.5 cm were cut and placed in a Petri dish and poured into Yeast Nitrogen Base (Difco), 0.657 culture fluid solution. g per 100 ml of distilled water The gels are immersed to about half their height After a three-fold sterilization at 100 ° C in steam (1 hour), a culture of Cryptacoiccus luteolus CCY 17-6 ^ 2b is inoculated onto the gel surface and incubated at 23 ° C. ° C for 48 hours The multiplied C. luteolus biomass on a stekutemic gel (0.21 grams) was used as an inoculum on the production soil (2 L) consisting of 0.8% corn steep liquor, 0.8% corn starch, 0 ° C. , 2% ammonium sulphate and 0,3% potassium dihydrophosphate in water (pH

6.1). Cultivation was carried out for 85 hours at 25 ° C. After the cultivation was complete, C. luteolus biomass was removed by centrifugation (31000 grams, 5 min) and the supernatant was concentrated in a vacuum evaporator. The obtained crude α-amylase enzyme had a total activity of 34.3 U and a specific activity of 0.081 U / mg.

Example 1 a d 2

The procedure was as in Example 1, except that the Cryptococcus luteolus CCY 17-6-2b strain was cultured on a culture medium containing 2% corn starch at 20 ° C for 120 hours and pH 4.7. Instead of concentration in a vacuum evaporator, the enzyme preparation is further processed by the affinity method using sintered starch (AO No. 157 955). The purified enzyme obtained had a specific activity of 11.4 U / mg. Example 3

The procedure was as in Example 1, except that the culture was carried out on a nutrient broth containing 3% corn starch at 38 ° C for 60 hours and pH 6.8. The .alpha.-amylase enzyme is obtained by precipitation with ammonium sulfate (680 g / l). The obtained enzyme preparation had a specific activity of 0.11 U / mg.

The importance of the invention lies in the use of α-amylase in the food industry (baking and brewing industries), the preparation of syrups and the like) and in the textile industry. The amylase hydrolyzate is used to prepare atypical sweeteners and pure ollgosaccharides (maltose, maltotriose, maltotetraose) as well as D-glucose for use in sacharide chemistry and in clinical practice.

Claims (3)

SUBJECT Process for producing amylase enzyme on a liquid nutrient medium comprising assimilable carbon sources, complex nitrogen sources and mineral nutrient salts, characterized in that the Cryptococcus l-uteoluls CCY 17-6-2b strain is cultivated on a nutrient medium containing 0.8 to 0.8 3 wt. % corn starch at 20 to 38 ° C for 48 to 120 hours at pH 4.7 to 6.8, wherein the α-amylase enzyme is obtained by evaporating the growth soil, or by precipitation with ammonium sulfate, or by affinity purification on a cross-linked starch.