SU600175A1 - Method of biological synthesis of biologically active substance - Google Patents

Description

one

The invention relates to the production of enzyme preparations, such as p-galactosidase, from microorganisms, concerns the method of biosynthesis of biologically active substances and can find wide application in the food industry related to the processing of milk and waste of the dairy industry, as well as for medical purposes.

There are known methods for biosynthesis of biologically active substances, namely, p-galactosidase (lactase, p - D-galactoside galactic hydrolase KF 3.2.1.23), which breaks milk sugar (tact) into glucose and galactose.

The active producers of p-galactosidase were revealed, among them the culture of the yeast Fabospoga fragilis (B 100 ml of medium produced 150-350 units of enzyme, in 1 g of dry yeast - up to 200 units), Escherichia coli, isolated from colibacterin (in 100 ml of medium formed up to 40 units of ip-galactosidase, and in 1 g of bacteria - up to 540 units of enzyme). There are other strains.

However, such strains for the implementation of supersynthesis are so demanding on cultivation conditions (chemostat, cultivated at low population densities) that they have not found application in industry.

In the overwhelming majority of cases, to obtain maximum product yield

perform special parametric or individual regulation of the biosynthesis process.

A more effective method of biosynthesis of a biologically active substance is known, according to which biosynthesis is regulated informationally by introducing exogenous information at a given moment, i.e. mixing the culture-producer with the information carrier. If, according to a known method, sensitive bacteria are infected with a bacteriophage carrying information on the synthesis of a given product, then in a short time there is a tremendous increase in this substance, reaching thousands of times.

This method of biosynthesis provides a sharp increase in product yield. However, there are also significant technological complications associated with the additional operating time of 1 formate (in this case, a bacteriophage). This is fraught with considerable technical difficulties, so great that the method practically becomes impracticable.

The aim of the invention is to increase the yield of the final product while simplifying the process.

For this, a lysogenic culture is used as an information carrier, while

the last before mixing with the culture of the producer is subjected to heating.

The proposed method is as follows. Producer of information, in this case lysogenesis culture - strain of Escherichia coli CA 5013 y. placsCiSS was first grown at 30 ° C, cooled to 4-6 ° C, and thermal induction was carried out at 42-43 ° C for 20 minutes. Then lysogenic culture immediately after thermal induction is mixed with the recipient strain. The recipient in this case is a phage-susceptible strain of Escherichia coli, (for example, Escherichia coli strain AB 259 Hfr3000. Lysocene culture after thermal induction is mixed in a ratio of 1: 3-1: 30, then continue incubation at 37 ° C under conditions of x strong aeration before the termination of the enzyme biosynthesis. After the termination of the process activity, p-galactosidase in the lysate reaches up to 10,000 units. It has been established that if there is 1 phage corpuscle per cell of Escherichia -coll, 5-10 times less than asset This is due to the fact that while the phage l is developing in a part of the cells, another part of the non-infected cells continues to grow and multiply and by the time of maturation and release from the cells portions of phage corpuscles for him there is a new generation of young cells that are infected with phage. This goes through several cycles, resulting in the accumulation of p-gala-whozidase.

According to the proposed method, 15-18 mg of protein are obtained (Z-galactosidase in I l of lysate or 10,000 units of enzyme in 1 ml of lysate. The conversion of units of p-galactosidase activity to the amount of enzyme protein is based on the data provided by Robinson (Biotechn. bioeng., 1974, No. 16, p. 1103).

Both cultures are grown in the environment aminoieptid with 0.14 M NaCl in a 1: 1 ratio. Such a medium is optimal compared with other nutrient media studied.

The resulting ratio of lysogeny and recipient cultures in ratios 1: 3-1: 30 is preferable compared to other ratios, since this results in maximum biosynthesis of the enzyme. According to the above data, it can be concluded that the indicators of the results of the synthesis by the proposed method are many times higher than the level of synthesis of the currently existing industrial producers of p-galactosidase, for which only tens of units of the enzyme are obtained.

Example. Lysogenic culture - Eseherichia coli CA strain 5013 l placa Ci857 on the medium aminopeptide with 0.14 M NaCI in a ratio of 1: 1 at 30 ° C to a density of 1X10 cells in 1 ml. Then the culture is cooled to 4-6 ° C and the thermal induction of the lysogenic culture is conducted at 42-43 ° C for 20 minutes. By this time, Escherichia coli AB 259 nfr 3000 producing culture is grown on an aminopeptide medium with 0.14 M NaCI in a 1: 1 ratio at 37 ° C to a density of 1X10® cells in 1 ml and mixed in a thermally induced lysogenic culture in a ratio of 10: 1 The process continues at 37 ° C for 18 hours. During this time, 10,000-15,000 units of the enzyme (but Purdy, Jacobou and Monot) accumulate in 1 ml of culture medium, which corresponds to 15-18 mg of protein rgalactosidase in 1 liter of lysate.

F o r M u l a n 3 o b r e e i u

Claims (2)

1. A method of biosynthesis of a biologically active substance, which involves mixing the producer culture with a carrier m of information, which is used in order to increase the yield and simplify the process of obtaining the target product, as the carrier of information lysogenic culture, while the latter is heated prior to mixing with the producer culture. 2. A method according to claim 1, characterized in that the heating is carried out in the range of 40-45 ° C.