CN1899599A - Method for preparing Nosiheptide premixed agent - Google Patents

Abstract

The invention belongs to the field of biopharmaceuticals, and relates to a method for preparing a medicinal feed additive, in particular to a method for preparing a sulfur-containing polypeptide antibiotic nosiheptide premix. The invention prepares nosiheptide-producing bacteria through genetic engineering, carries out nosiheptide fermentation, separates and extracts, adds auxiliary materials, dilutes step by step, mixes and mixes well, and prepares nosiheptide premix. The test results show that the method can improve the oxygen utilization efficiency of the active streptomyces and make the bacteria grow vigorously, so as to achieve the purpose of increasing the yield of nosiheptide and reducing the production cost.

Description

A kind of method for preparing nosiheptide premix

technical field

The invention belongs to the field of biopharmaceuticals, and relates to a method for preparing a medicinal feed additive, in particular to a method for preparing a sulfur-containing polypeptide antibiotic nosiheptide premix.

Background technique:

Feed safety is the basic point of green and environmentally friendly food. my country has a vast territory and a large population. With the acceleration of reform and opening up and the improvement of people's living standards, the demand for livestock, poultry and aquatic products is also increasing. In addition, my country is a large agricultural country, and the export volume of poultry and livestock products is very large, but the problem of drug residues is very serious. Therefore, the research and development of green feed additives is very necessary for the protection of people's health and the development of my country's agricultural economy. In July 2001, the Ministry of Agriculture issued the notice of "Specifications for the Use of Feed Drug Additives", and listed 33 kinds of feed drug additives that meet the specifications, and Nosiheptide is one of them. It is a new type of non-absorbable feed antibiotic.

The new veterinary drug Nosiheptide (also known as Nosiheptide, English name Nosiheptide) is a product of bioengineering, produced by the fermentation of Streptomyces actuosus. It is a sulfur-containing polypeptide antibiotic. Its structure is a polypeptide synthesized by 12 amino acids and derivatives, with a molecular weight of 1222 . Nosiheptide is a yellow crystal, which is difficult to dissolve in water. Its structure (Merck index, fourth edtion) is shown in Figure 7. The molecule consists of five thiazole rings, a pyridine ring, an indole ring, a threonine and a glutamic acid, and the side chain is a dehydroalanine. Nosiheptide has a strong inhibitory effect on Gram-positive bacteria, and its mechanism of action is to inhibit the elongation factors Tu and G in the translation process and inhibit the growth of Gram-positive bacteria. Simultaneously inhibit the synthesis of guanosine three and four phosphatases.

Nosiheptide is a compound discovered abroad in 1961. In the 1970s, the French Rumplock Company conducted research on it. In the 1980s, it was developed into a new type of non-absorbable feed antibiotic by Mitsubishi Corporation of Japan. It was released on December 25, 1987. Approved by the Japanese government as a statutory feed additive, listed in the Japanese Feed Safety Law, its trade name is PrilnofaX (Hess & Clark). Due to its non-residual nature in the body, it is currently widely used in the European Community, North America, Japan and other countries

As a feed additive, nosiheptide has the following characteristics: 1. Animal-specific antibiotics do not cause cross-resistance. 2. No residue in animal body, no pollution to food. 3. The addition amount is small (5-10ppm), and the growth-promoting effect is obvious (weight gain 8-10%). And increase feed remuneration. 4. It has a wide range of applications and can be used for poultry, livestock, fish, etc.

In 1988, Nosiheptide was developed and researched by Shanghai Medical University, and in June 1998, it was awarded Nosiheptide New Veterinary Drug Certificate (Class III) (raw material drug and premix) by the Ministry of Agriculture. According to the test and identification of nosiheptide samples by the China Veterinary Drug Control Institute, the product quality is completely consistent with similar foreign products. In 2002, Zhejiang Hangzhou Huineng Biotechnology Co., Ltd. cooperated with Fudan University to industrialize Nosiheptide. Among them, Nosiheptide-producing bacteria S.actuosus Z13 is the production strain, and the trade name is "Nuonon". The current annual sales output value is 70 million. And it is widely used in poultry, livestock and aquatic products to obtain good social benefits. However, looking at domestic and foreign literature, there is no report on the preparation method of nosiheptide premix.

Contents of the invention

The object of the present invention is to provide a method for preparing nosiheptide premix. Especially the method for preparing nosiheptide premix of sulfur-containing polypeptide antibiotics.

The inventive method comprises the steps:

1. Preparation of nosiheptide-producing bacteria Streptomyces actuosus by genetic engineering

Based on the research of the known nosiheptide-producing bacterium S.actuosusZ13, the invention adopts genetic engineering technology to change the metabolism of the nosiheptide-producing bacterium Z13 and increase its output.

First, adopt DNA recombination technology to construct the recombinant DNA containing the gene vhb of Vitella hyaline hemoglobin (VHb) (see accompanying drawings 1 and 2 for genetic engineering operations), and transfer the recombinant DNA containing the vhb gene into Z13 by transforming and combining transfer methods. , obtain engineering streptomyces streptomyces actuosus, (engineering bacterium code Z14, Z15). In the case of insufficient oxygen supply in industrial production, due to the large expression of VHb protein, the oxygen utilization efficiency of active Streptomyces. production cost purposes.

Second, the present invention adopts genetic engineering technology to construct the recombinant DNA containing the gene sam of S-adenosylmethionine synthetase SAMs (gene engineering operation is shown in accompanying drawing 3,4), will contain sam by transforming and combining transfer method The recombinant DNA of the gene was transferred into Z13 to obtain engineering Streptomyces actuosus engineering bacteria (engineering bacteria code Z16, Z17). Through the expression of SAMs protein, the sulfur metabolism of active Streptomyces.actuosus Z13 is changed, and the production of S-adenosylmethionine is increased, so as to achieve the purpose of increasing the production of nosiheptide.

The 3rd, the present invention adopts genetic engineering technology to construct the recombinant DNA containing two genes of vhb and sam, (genetic engineering operation sees accompanying drawing 5,6), will contain the recombinant DNA of vhb and sam two genes by transformation and combination transfer method Transform into Z13 to obtain engineering Streptomyces actuosus (engineering bacteria code Z18, Z19), and change the metabolism of active Streptomyces through the expression of VHb and SAMs proteins to achieve the purpose of increasing the production of nosiheptide.

2. Nosiheptide fermentation;

Nosiheptide fermentation medium is composed of soybean powder, glucose, inorganic salts, etc., the optimal temperature is controlled at 26-30°C, the pH is neutral, and the fermentation period is 9-10 days.

3. Separation and extraction of nosiheptide:

Use the solvent method to extract nosiheptide in the bacteria, measure the volume of the fermentation broth, add an equal volume of alcohol, homogenize, centrifuge, collect the supernatant, concentrate, extract with n-butanol, concentrate and evaporate to dryness, add dioxane Dissolve, then add water to precipitate nosiheptide, wash the precipitated nosiheptide with water, and dry. The content was determined by HPLC and the biological activity was detected according to the Veterinary Pharmacopoeia.

4 Preparation of nosiheptide premix:

Take nosiheptide, add starch, soybean cake powder, rice bran powder, corn flour, bran, rice protein powder, stone powder (CaCO ₃ ), zeolite powder and other auxiliary materials, dilute step by step, mix well, and make 0.25%-10 % Premixes of different specifications. The content was determined by HPLC and the biological activity was detected according to the Pharmacopoeia.

Description of drawings

Fig.1 Construction of vhb transformation expression plasmid

Figure 2, the construction of the vector containing vhb integration

Fig.3 Construction of expression plasmids containing sam transformation

Figure 4, construction of sam-containing integration vector

Figure 5, construction of transformation vectors containing vhb and sam

Figure 6, construction of integrated vectors containing vhb and sam

Figure 7, Nosiheptide structure diagram.

Detailed ways

Example 1

Nosipeptide-producing bacteria Z13 was inoculated in the seed tank, grown for 40-48 hours, inserted into a 30-ton fermenter with 5% inoculum, with 1:1 ventilation, controlled temperature at 28-30°C, and stirred at 160-180 rpm According to the pH, sugar content and thallus morphology, the feed is divided into 9 days, and 20 tons of material are discharged, and the fermentation unit per liter reaches 2.3 grams. After extracting 23.46 kg of nosiheptide (purity 85%); add auxiliary materials such as starch, soybean cake powder, rice bran powder, corn flour, bran, rice protein powder, stone powder (CaCO ₃ ), zeolite powder, and gradually dilute, Mix well to obtain 7.98 tons of premix (0.25% specification).

Example 2,

Nosiheptide-producing bacterium Z14 was inoculated in the seed tank, grown for 40-48 hours, inserted into a 30-ton fermenter with 5% inoculum, with 1:0.5 ventilation, controlled temperature at 28-30°C, and stirred at 160-180 rpm According to the pH, sugar content, and thallus morphology, feed was fed, cultivated for 9 days, and 20 tons of material were discharged, with a fermentation unit of 3.2 grams per liter. After extraction, 32.6 kg of nosiheptide (purity 85%) were obtained; starch, soybean cake powder, bran, rice bran powder, rice protein powder, stone powder (CaCO ₃ ), zeolite powder, etc. were added, diluted step by step, mixed well, and obtained 11.08 tons of premix (0.25% specification).

Example 3,

Nosiheptide-producing bacterium Z15 was inoculated in the seed tank, grown for 40-48 hours, inserted into a 30-ton fermenter with 5% inoculum, with 1:0.5 ventilation, controlled temperature at 28-30°C, and stirred at 160-180 rpm According to the pH, sugar content, and bacterium morphology, feeding was carried out for 9 days, and the output was 21 tons, and the fermentation unit per liter reached 4.2 grams. After extraction, 44.982 kg (purity 85%) of nosiheptide was obtained; starch, soybean cake powder, rice bran powder, bran, corn flour, stone powder (CaCO ₃ ), zeolite powder, etc. were added, diluted step by step, mixed well, and prepared Mixture (0.5% specification) 7.65 tons.

Example 4,

Nosiheptide producing bacterium Z16 was inoculated in the seed tank, grown for 40-48 hours, inserted into a 50-ton fermenter according to 5% inoculum, with 1:0.8 ventilation, controlled temperature at 28-30 ° C, and stirred at 160 rpm. Among them, according to the pH, sugar content, and thallus morphology, the material was fed, cultivated for 9 days, and the output was 35 tons, and the fermentation unit per liter reached 4.5 grams. After extraction, 80.33 kg of nosiheptide (purity 85%) was obtained; rice bran powder, bran, corn flour, rice protein powder, stone powder (CaCO ₃ ), zeolite powder, etc. were added, diluted step by step, mixed well, and a premix was obtained (0.5% specification) 13.656 tons.

Example 5,

Nosiheptide-producing bacterium Z19 was inoculated in the seed tank, grown for 40-48 hours, inserted into a 50-ton fermenter according to 5% inoculum, with 1:0.5 ventilation, controlled temperature at 28-30°C, and stirred at 160 rpm. Among them, according to the pH, sugar content, and thallus morphology, the material was fed, cultivated for 9 days, and the output was 35 tons, and the fermentation unit per liter reached 5.0 grams. After extraction, 89.25 kg of nosiheptide (purity 85%) was obtained; rice bran powder, bran, corn flour, rice protein powder, stone powder (CaCO ₃ ), zeolite powder, etc. were added, diluted step by step, mixed well, and a premix was obtained (5% specification) 1.517 tons.

Claims (5)

1, a kind of method for preparing Nosiheptide premixed agent is characterized in that comprising the steps:

1) genetic engineering prepares nosiheptide generation bacterium;

2) nosiheptide fermentation;

3) separation and Extraction nosiheptide;

4) get above-mentioned nosiheptide, add adjuvant, stepwise dilution mixes and mixes, prepares Nosiheptide premixed agent thoroughly.

2, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that adopting in the described step 1 the DNA recombinant technique, structure contains the recombinant DNA of the gene vhb of Vitreoscilla hemoglobin, by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain the vhb gene, obtain engineering streptomycete Z14, Z15;

Structure contains the recombinant DNA of the gene sam of S-adenosylmethionine synzyme SAMs, by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain the sam gene, obtains engineering streptomycete Z16, Z17;

Build the recombinant DNA that contains vhb and two genes of sam,, obtain engineering streptomycete Z18, Z19 by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain vhb and two genes of sam.

3, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 2 wherein the nosiheptide fermentation medium contain analysis for soybean powder, glucose and inorganic salt composition, the control temperature is 26-30 ℃, pH be a neutrality, fermentation period is 9-10 days.

4, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 3 wherein adopts the solvent method to extract endobacillary nosiheptide, fermentation liquid is measured the volume number, add equal-volume ethanol, homogenate, centrifugal, collect supernatant, concentrate, with n-butanol extraction, concentrate evaporate to dryness, add the dioxane dissolving, add the water precipitation nosiheptide again, the water washing and precipitating thing, content and detection of biological activity are measured in oven dry.

5, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 4 wherein the content of nosiheptide be 0.25%-10%, described adjuvant comprises starch, soybean cake powder, bran powder, Semen Maydis powder, wheat bran, rice protein powder, CaCO ₃And zeolite powder.

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