RU2272838C1 - Strain of bacterium bacillus macerans bs-04 as producer of pectinases, protease, xylanase and amylase - Google Patents

Abstract

FIELD: biotechnology, microbiological industry, biochemistry.

SUBSTANCE: invention relates to producing enzymes and represents a new strain of Bacillus macerans BS-04 as a producer of pectinases, protease, xylanase and amylase. This strain provides preparing the broad complex of enzymes and characterized by short cycle of growth in combination with enhanced accumulation of biomass.

EFFECT: valuable properties of strain.

1 tbl, 4 ex

Description

The invention is a new strain of Bacillus macerans BS-04 and relates to the microbiological industry that produces enzyme preparations, and can be used to obtain enzyme-containing drugs used to treat and prevent diseases associated with digestive disorders; and in the preparation of preparations used to increase the yield and improve the quality of vegetable and fruit juices and mashed potatoes, clarified fruit and berry wine, essential and vegetable oils, tea, in the processing of difficult-to-digest vegetable feeds and in obtaining biological additives for agriculture.

Known Clostridium pectinofermentas pcs. 15 - anaerobic producer of macerating enzymes intended for use in the flax processing industry (1). Clostridium pectinofermentas pcs. 15 synthesizes pectate-transeliminase (0.55 units / ml), exo-polygalacturonase (2.5 units / ml), pectin esterase (0.13 units / ml), xylanase (0.96 units / ml) and does not synthesize endo-polygalacturonase and cellulase. A significant disadvantage of this strain is the low level of enzyme biosynthesis.

A known strain of Bacillus circulans pc. 31 CMPM B-1741 - producer of macerating enzymes (2). Bacillus circulans pcs.31 is an optional anaerobic. Optimum cultivation conditions: pH 8.1–8.3; temperature 37-42 ° C; the cultivation time is 36 hours. The producer synthesizes pectolytic enzymes: pectate-transeliminase from 2 to 10 units / ml, endo-polygalacturonase - 25 units / ml. Significant disadvantages of the strain Bacillus circulans pcs.31 are: low activity of synthesized enzymes and a long period of cultivation of the producer.

The known bacterial strain Bacillus circulans VKMP-B-5622 is a producer of a complex of pectolytic enzymes and xylanase (3). The strain synthesizes enzymes: pectate-transeliminase - on a medium containing beet pulp, from 3800 to 6700 u / ml; and on a medium containing potato starch from 10,000 to 25,000 units / ml; endo-polygalacturonase from 32.57 to 87.7 units / ml; exo-polygalacturonase from 12.0 to 36.77 units / ml and xylanase to 30.8 units / ml. The strain is cultured for 10-18 hours at a pH of 7.8-8.6 and a temperature of 36-45 ° C.

The closest analogue (prototype) to the claimed strain is a strain of Bacillus macerans CMPM B-2692 - producer of pectolytic enzymes (4), which under conditions of deep cultivation forms endo-polygalacturonase with an activity of 6.7 units / ml, exo-polygalacturonase with an activity of 5, 3 units / ml and pectate-transliminase with an activity of 9000 units / ml. The cultivation of the producer is carried out at a temperature of 32-35 ° C, pH 6.8-7.2 for 18-20 hours. A significant drawback of this strain is the insufficiently high activity of pectate-transliminase and a narrow complex of synthesized enzymes.

The aim of the present invention is to obtain a highly active strain of the facultative anaerobic bacterium Bacillus macerans BS-04 - capable of synthesizing a wide range of enzymes with an increased synthesis of pectinases, namely pectate and pectin lyases, endo- and exo-polygalacturonases, pectin esterases; and additional synthesis of protease, xylanase, amylase, and characterized by a short growth cycle with increased biomass accumulation.

The inventive strain of Bacillus macerans BS-04 is isolated as a result of multiple screening of the original strain of Bacillus macerans CMMP B-2692 and isolation of monospore variants. The strain Bacillus macerans BS-04 has the following cultural, morphological and physiological characteristics.

Cultural and morphological features

Cells are straight, rod-shaped, 2.0-5.0 × 0.9-1.0 μm, motile, gram-positive, are found in long and short chains. Spores are ellipsoidal, without exosporium, 2.0-2.4 × 1.0-1.2 μm, located centrally or subterminally.

On potato agar:

- on the first day, growth is good, the colonies are light beige, round, the middle is rough, matte, the edges are uneven, shiny, the size of the colonies is from 1 to 4 mm in diameter. The pigment does not diffuse on Wednesday;

- on the second day, the colonies are light beige, round, the middle is rough, matte, the edges are uneven, shiny, the size of the colonies is from 1 to 6 mm in diameter;

- on the sixth day, the colonies are from light beige to beige, shiny, uniform, round, the edges are uneven, the size of the colonies is from 1 to 6 mm in diameter.

On wort agar:

- on the first day the colonies are light beige with a lighter and denser middle, matte, rough, the edges are even, the size is from 0.5 mm to 5 mm;

- on the second day, the colonies are light beige with a denser convex, shiny, opaque middle, the edges of the colony are dull, rough, opaque, uneven, the size of the colonies is from 1 mm to 10 mm;

- on the third day, the colonies are light beige with a lighter and denser, slightly convex middle. All colonies are shiny, translucent, the edges are uneven. Colony size from 1 mm to 11 mm.

On Capek’s environment with glucose:

- on the first day, the colonies are homogeneous, convex milky white, shiny, transparent, the edges are even, the size of the colonies is from 0.5 mm to 1.5 mm;

- on the second day, the colonies are heterogeneous light beige, the middle is shiny in the form of a button, the edges are uneven, matte, rough, the size of the colonies is from 1 mm to 4 mm;

- on the third day, the colonies are heterogeneous light beige, the middle is shiny in the form of a button, the edges are uneven, matte, rough, the size of the colonies is from 1 mm to 7 mm;

- on the sixth day, the colonies are heterogeneous, the edges are uneven, matte, rough; the middle is shiny in the form of a button, opaque, the size of the colonies is 0.2-1.2 mm, the color is light beige.

On Czapek’s environment with starch:

- on the first day, the size of the colonies is 0.01-0.5 mm, the colonies are shiny, opaque, uniform, convex, white, the edges are uneven;

- on the third day, the size of the colonies is 0.5-2.0 mm, the colonies are shiny, opaque, uniform, convex, milky white, the edges are uneven;

- on the fourth day, the colonies are 0.5-2.5 mm, shiny, convex, opaque, milky white, the center is denser (pin densification), the edges are uneven, deeply incised;

- on the sixth day, colonies from 0.5 to 5 mm, opaque, shiny, heterogeneous with a point seal in the center, the edges are dull, uneven.

On meat peptone agar:

- weak growth, dotted colonies, beige.

Physiological and biochemical characteristics of the strain

Attitude to carbohydrates: assimilates glucose, sucrose, xylose, arabinose, maltose and mannitol with the formation of acid and gas, hydrolyzes starch.

Forms catalase, acetone, crystalline dextrins. Nitrates are reduced to nitrites. Phenylalanine does not deamine; tyrosine does not cleave. Ammonia and hydrogen sulfide does not form. Hippurates do not hydrolyze. Gelatin dilutes 1 cm at the top. Milk peptones with the formation of a clot and gas. It grows on a medium with 0.001% lysozyme, does not grow on a medium with 5% sodium chloride.

It grows well on slices of carrots, potatoes, turnips, white cabbage, zucchini, pumpkin, apple (non-acidic varieties) and pears with complete maceration of the substrate.

On an environment with 1% sodium nitrate under anaerobic conditions, a delay in spore formation is noted.

Attitude to oxygen is an optional anaerobic. The minimum growth temperature is + 10 ° C, maximum + 45 ° C, optimal 37-42 ° C, pH 7.4-7.8.

The cultivation time under aerobic conditions is 10-18 hours, under conditionally anaerobic conditions - 18-30 hours.

The producer is subject to seasonal influences, therefore, in the spring and autumn periods, a decrease in enzymatic activity by 10-15% is observed.

The activity of pectin- and pectatliases (on the corresponding substrates) was determined by spectrophotometry of unsaturated products of the pectolysis reaction having a maximum optical density at a wavelength of 235 nm according to TU opt.5800665-2-91. The unit of activity is such an amount of enzyme that for 1 h at 40 ° C causes the formation of unsaturated reaction products, increasing the optical density by 0.1.

Endo-polygalacturonase activity (endo-PG) was determined by the viscometric method according to GOST 20264.3-81, exo-polygalacturonase activity (exo-PG) was determined by the titrometric method to increase the reducing groups according to GOST 264.3-81, pectin esterase activity was determined according to GOST 20264.3-81, proteolite activity (alkaline protease) was determined using sodium caseinate as a substrate according to GOST 20264.2-74, xylanase activity was determined according to TU 9291-035-34588571-2001, amylase activity was determined according to GOST 20264.4-89. The determination of biomass accumulation (cell titer) was determined by the method of limiting dilutions. The determination of macerating ability was carried out according to a test based on the determination of the change in the mass of the plant substrate as a result of the action of enzymes of the culture fluid of the claimed producer. The degree of maceration of plant substrates was determined by the decrease in the mass of the substrate treated with the enzyme in comparison with the control variants and was expressed as a percentage.

Examples of cultivation of a strain of Bacillus macerans BS-04

Example No. 1

The culture was grown on medium, g / l:

Ground beet pulp 20,0 Calcium chloride 0.9 Sodium carbonate (soda) 2.0 Disubstituted Ammonium Phosphate 2.6 Monosubstituted sodium phosphate 2.2 Corn extract 7.4 Tap water rest

The nutrient medium was poured into 100 ml into rocking flasks with a capacity of 750 ml and sterilized at an excess pressure of 0.1 MPa for 45 minutes. As seed, spores of the Bacillus macerans BS-04 strain were used, which are formed on the surface of mowed potato agar after inoculation and growth for 48 h at 40 ° C. After washing off from 1 jamb, 3 flasks were sown in an amount of 3% of the volume of the nutrient medium. Cultivation was carried out under aerobic conditions on a rocking chair with a rotational speed of 180-200 rpm at a pH of 7.6, a temperature of 40 ° C and for 10 hours

Example No. 2

Cultivation was carried out on the medium in accordance with example No. 1, with the exception of aeration, temperature and pH of the medium. Cultivation was carried out under conditionally anaerobic conditions. For this purpose, round flat flasks with a narrow neck were used. The nutrient medium was poured in an amount of 0.5 volume of the flask. Cultivation was carried out at a pH of 7.8, a temperature of 42 ° C, for 24 hours without stirring.

Example No. 3

Cultivation was carried out for 18 hours under conditions corresponding to the conditions of example No. 1. The composition of the nutrient medium differed from the composition of Example No. 1 with an additional content of 1% casein and 1% of Biotrin fodder yeast.

Example No. 4

The producer was grown on medium, g / l:

Potato starch 100.0 Amylosubtilin G3x with AC 550 u / g 0.37 Disubstituted ammonium citrate 30,0 Disubstituted sodium phosphate 3.0 Potassium phosphate monosubstituted 2.0 Magnesium sulfate 1,5 Calcium sulfate 1,5 Ferrous sulfate 0.02 Manganese Sulfate 0.00125 Copper sulfate 0.0075 Aminobenzoic acid 0.01 Distilled water rest pH of the medium 7.4

Potato starch, which is part of the nutrient medium, was preliminarily saccharized with amylosubtiline GZx with an AC of 550 u / g. A culture medium of 300 ml was poured into flasks with a capacity of 2 L and sterilized at 1.5 MPa. The cultivation was carried out under conditionally aerobic conditions at a pH of 7.4, a temperature of 37 ° C without stirring for 30 hours

At the end of producer cultivation, enzyme activity was determined in the supernatant obtained from the culture fluid by centrifugation at 5000 rpm for 10 min. Data on the results of the experiments are presented in the table.

The use of the proposed strain of Bacillus macerans BS-04 in comparison with the prototype has the following advantages:

1. Increased synthesis of pectatliase up to 60,000 units / ml (in the prototype up to 9,000 units / ml), pectin lyase up to 25,000 units / ml (in the prototype - not available).

2. A higher level of synthesis of endo-polygalacturonase activity: at a substrate pH of 4.0 - up to 9 units / ml, and at a substrate pH of 8.0 - up to 909 units / ml (in the prototype - 6.7 units / ml).

3. A higher level of synthesis of exo-polygalacturonase activity up to 15.1 u / ml (in the prototype - 5.3 u / ml).

4. The synthesis of pectin esterase activity reaches 57.84 units / ml (in the prototype is absent).

5. In addition to the above pectinases, the strain further synthesizes: alkaline protease (up to 0.76 units / ml), xylanase (up to 0.19 units / ml) and amylase (up to 83.2 units / ml).

6. Increased biomass accumulation (cell titer) reaches 2.7 × 10 ¹⁰ (data not available in the prototype).

Thus, the proposed strain has significant advantages compared with the prototype, which will allow for its industrial use to obtain highly active drugs with a wide range of enzymes for the medical and food industries, as well as for agriculture.

Bibliography

1. G. B. Bravova et al. Proceedings of the enzyme department of the All-Russian Research Institute of Synthesis Proteins, vol. 1, SENTI, 1972

2. USSR AS No. 1349250 "Bacillus circulans pcs. 31 TsMPM V-1741 - producer of macerating enzymes", 1985

3. RF patent No. 2072691 "Bacillus circulans bacterial strain - producer of a complex of pectolytic enzymes and xylanase", priority 1994

4. USSR AS No. 1162224 "Strain of Bacillus macerans TsMPM V-2692 - producer of pectolytic enzymes", 1984

Claims (1)

The strain Bacillus macerans BS-04 is a producer of pectinases, proteases, xylanases and amylases.