RU2307159C1 - BACTERIUM Azotobacter chroococcum 12A STRAIN AS PRODUCER OF POLY-3-OXYBUTYRATE AND COPOLYMER OF POLY-3-OXYBUTYRATE WITH 3-OXYVALERATE - Google Patents

Abstract

FIELD: biothechnology.

SUBSTANCE: Claimed strain is cultivated on nutrient medium containing sugars as main carbon source for poly-3-oxybutyrate synthesis and valeric acid as add carbon source for synthesis of copolymer thereof. Target products are obtained with yield of 30-90%.

EFFECT: new large scale method for synthesis of poly-3-oxybutyrate and copolymer of poly-3-oxybutyrate with 3-oxyvalerate.

3 ex

Description

The invention relates to the field of biotechnology and is a new strain of Azotobacter chroococcum used to produce a thermoplastic, biodegradable and biocompatible polymer of poly-3-hydroxybutyrate (PHB) and a copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate (WSP), which can be used in medicine - biodegradable and biocompatible surgical threads, plates, films for covering wounds, matrices for drugs of prolonged action with an adjustable release time, etc .; in industry, the inclusion of a biopolymer in other non-biodegradable polymers, packaging and disposable materials; and also in agriculture - systems for the slow release of fertilizers and agrochemicals.

Known producers of polyhydroxyalkanoates (POA) belonging to various bacterial genera Azotobacter, Alkaligenes, Methylobacterium (RF Patent No. 2051967, CL 12/04, 1992, RF Patent No. 2123048, CL 12/7/62, 1993) . They have high-tech indicators, such as high content of PHB and WSP in cells, biomass yield, biomass synthesis rate and biopolymer accumulation. However, they grow mainly on gaseous carbon sources, most strains are obtained by genetic means, which can lead to a loss of ability to synthesize the target product, the maximum polymer yield is usually 65-75%.

As a prototype of the selected strain Azotobacter chroococcum 7B - producer of PHB that can use carbohydrates as a carbon source. The yield of the target product is 70-90% (RF Patent No. 2194759, class C12P 7/62, C12N 1/20, 2001).

The objective of the invention is to obtain a new stable strain of bacteria capable of high-yield synthesis of PHB, as well as PHBV, which allows to obtain biopolymers with different physicochemical properties and thereby expands the scope of their application in various fields.

This problem is solved by a new strain of Azotobacter chroococcum 12A, which is a superproducer of PHB, as well as WSP. The strain is stored in the collection of the A.N. Bach Institute of the Russian Academy of Sciences. The strain isolated from sod-podzolic soil of the Moscow region.

The strain has the following characteristics:

Cultural and morphological features

Large oval-shaped cells, gram-negative, 2.0-2.5 × 3.5-5.0 μm in size with a fine-grained cytoplasm and compact nucleoid. Young cells have peritrichous flagella, as well as fimbriae, capable of movement. With age, the culture becomes polymorphic, the cells join together in chains, form filamentous forms and sarcin-like packets. Granularity appears in the cell cytoplasm associated with the accumulation of granules of the reserve compound of poly-3-hydroxybutyrate. The resting stage is characterized by the formation of cysts and mucous capsules. The colonies on Ashby's agar medium are translucent, whitish, mucosa-wrinkled, and eventually become brown-black.

Physiological and biochemical characteristics.

Relation to carbon sources - uses glucose, sucrose, fructose, galactose, maltose, mannitol, starch, methanol, ethanol, acetic, citric, propionic and valerianic acids.

Obligate aerob.

Attitude to nitrogen sources - fixes atmospheric nitrogen, uses nitrate nitrogen.

It does not dilute gelatin, it does not restore nitrates.

It grows at a temperature of +10 to + 40 ° C, the optimum growth in the range of 28-32 ° C.

The pH range at which growth is possible from 5.0 to 8.6 with an optimum of 7.2-7.6.

The strain produces up to 90% of PHB and WSP.

The optimum biosynthesis of PHB at 30 ° C and a pH of 7.2.

To obtain PHB, the Azotobacter chroococcum 12A strain is grown under aerobic conditions on a nutrient medium containing carbohydrates as a carbon source and mineral salts. And to obtain a WSP, an additional carbon source, an organic acid salt, is added to the main source of carbon - carbohydrates. After two days, the biomass yield is 2-12 g / l (dry water), and the content of PHB or WSP is 30-95%.

The invention is illustrated by examples.

Example 1

The strain Azotobacter chroococcum 12A is maintained on Ashby nutrient medium containing (g / L: K ₂ NRA ₄ - 0.2; MgSO ₄ - 0.2; NaCl - 0.2; Na ₂ MoO ₄ · 2H ₂ O - 0.006; CaCO ₃ - 5.0; sucrose - 20; agar - 20). Cultivation is carried out in flasks on a rocking chair (180 rpm) at a temperature of 28-30 ° C for 48 hours. The composition of the nutrient medium (g / l): MgSO ₄ · 7H ₂ O - 0.4; FeSO ₄ · 7H ₂ O — 0.01; Na ₂ MoO ₄ · 2H ₂ O - 0.006; Na citrate - 0.5; CaCl ₂ - 0.1; K ₂ HPO ₄ · 3H ₂ O - 1.05; KN ₂ RO ₄ - 0.2; sucrose-40. At the end of cultivation (48 hours), the biomass concentration is 9.7 g dry.v. / L, and the PHB content determined by the Brandl method (Brandl H., Gross R., Lenz R., Fuller R. Plastics from bacteria and for bacteria: poly (β-hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters // Adv. Biochem. Eng. Biotechnol, V.41. P.77-93, 1990), 85%.

Example 2

The method is carried out as in example 1, but to obtain a WSP, an additional carbon source is used - valerianic acid 1.0 g / L. At the end of cultivation (48 hours), the biomass concentration is 4 g dry.v. / L, and the WSP content determined using the Brandl method is 70%. The molar percentage of inclusion of 3-hydroxyvalerate in the polymer chain is 10%.

Example 3

The method is carried out as in example 1, but to obtain a WSP, an additional carbon source is used - valerianic acid 2.0 g / L. At the end of cultivation (48 hours), the biomass concentration is 2.5 g dry.v. / L, and the WSP content determined using the Brandl method is 62%. The molar percentage of inclusion of 3-hydroxyvalerate in the polymer chain is 18%.

Claims (1)

The bacterial strain Azotobacter chroococcum 12A is a producer of poly-3-hydroxybutyrate and a copolymer of 3-hydroxybutyrate with 3-hydroxyvalerate.