阅读说明：本技术 一株赤红球菌及其用于烟酰胺生产的方法 (Rhodococcus ruber and method for producing nicotinamide by using same ) 是由 李自云 邵树强 于 2020-04-01 设计创作，主要内容包括：本发明属于生物技术领域,具体地涉及一株赤红球菌及其用于烟酰胺生产的方法；包括赤红球菌催化剂的制备和水合反应等步骤,以赤红球菌发酵产生的腈水合酶为催化剂,以纯水为反应体系,通过流加或批次添加的方式,将3-氰基吡啶催化转化为烟酰胺；本发明提供的产腈水合酶的赤红球菌遗传稳定性好,培养成本低,产生的腈水合酶酶活力高,可直接利用该菌株将3-氰基吡啶转化为烟酰胺。催化反应时,以纯水为反应体系,反应液终反应浓度可达400g/L以上,避免引入外源杂质,同时有利于提取分离操作,工业应用具有优势。(The invention belongs to the technical field of biology, and particularly relates to Rhodococcus ruber and a method for producing nicotinamide by using the Rhodococcus ruber, which comprises the steps of preparation of a Rhodococcus ruber catalyst, hydration reaction and the like, wherein nitrile hydratase generated by fermentation of the Rhodococcus ruber is used as the catalyst, pure water is used as a reaction system, and 3-cyanopyridine is catalytically converted into nicotinamide by a fed-batch or batch adding mode.)

1. A method for producing nicotinamide by Rhodococcus ruber is characterized in that: the method comprises the following process steps:

(1) preparation of a rhodococcus ruber catalyst:

seed culture: adding culture medium 40-90% of the seeding tank volume into the seeding tank, and adjusting pH to 6.5-8.0 with sodium hydroxide; sterilizing at 125 ℃ for 25-35 minutes at 118-;

fermenting strains: adding culture medium 40-90% of the volume of the fermentation tank into the fermentation tank, and adjusting pH to 6.5-8.0 with sodium hydroxide; sterilizing at 125 ℃ for 25-35 minutes at 118-;

(2) hydration reaction:

removing culture medium components from fermentation broth after fermentation culture, adding the fermentation broth into a reaction kettle, diluting thalli by pure water, controlling the liquid loading amount to be 60% of the volume of the reaction kettle, controlling the reaction temperature to be 20-60 ℃, slowly adding 3-cyanopyridine in a flowing manner for reaction, stopping adding the 3-cyanopyridine in the flowing manner when the reaction concentration reaches above 400 g/L, tracking the residual amount of the 3-cyanopyridine by liquid chromatography to be less than 0.01, and finishing the reaction to obtain the nicotinamide.

2. The method for producing nicotinamide by using rhodococcus ruber according to claim 1, wherein the method comprises the following steps: the culture medium in the seed culture step comprises the following components in percentage by mass: 0.2-1% of yeast extract, 0.5-2% of glucose, 0.1-2% of sodium chloride, 0.02-1% of magnesium sulfate heptahydrate, 0.02-1% of potassium dihydrogen phosphate, 0.02-1% of dipotassium hydrogen phosphate and 0.01-0.5% of defoaming agent.

3. The method for producing nicotinamide by using rhodococcus ruber according to claim 1, wherein the method comprises the following steps: the culture medium in the strain fermentation step comprises the following components in percentage by mass: 0.2-1% of yeast extract, 0.5-2% of glucose, 0.1-5% of corn steep liquor, 0.1-2% of ammonium sulfate, 0.02-1% of magnesium sulfate heptahydrate, 0.02-1% of potassium dihydrogen phosphate, 0.02-1% of dipotassium hydrogen phosphate and 0.01-0.5% of defoaming agent.

4. The method for producing nicotinamide by using rhodococcus ruber according to claim 1, wherein the method comprises the following steps: the strain used in the step (1) is Rhodococcus ruber; the Rhodococcus ruber is preserved in China general microbiological culture preservation management center, and the preservation date is as follows: 28/10/2019, accession number: CGMCC No. 18759.

5. A method of using Rhodococcus ruber for nicotinamide production according to any of claims 1-4, characterized in that: the rhodococcus ruber cell is in a short rod shape, and the bacterial colony is round, orange yellow, gram positive, catalase positive and oxidase negative.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to rhodococcus ruber and a method for producing nicotinamide by using the same.

Background

Niacinamide (niacinamide), also known as nicotinamide, vitamin B3, vitamin PP, is a white crystalline powder; no or almost no odor, bitter taste; it is slightly hygroscopic. It is easily soluble in water or ethanol, and soluble in glycerol. The clinical medicine is vitamin B group, and can be used for preventing and treating pellagra, stomatitis, glossitis, etc. It is a nutrient essential for mammals and has better water solubility than nicotinic acid. Niacin and nicotinic acid are commonly used in most cases, and nicotinic acid also produces nicotinamide in animals. Nicotinamide is used as a nutritional additive in cosmetics. In addition, it can be used as food and feed additive. Has wide market prospect.

The production of nicotinamide is mainly divided into chemical and biological methods. The chemical synthesis method mainly produces nicotinamide and nicotinic acid by hydrolyzing 3-cyanopyridine, the reaction selectivity is poor, and the nicotinic acid content in the nicotinamide product is relatively high. Compared with a chemical method, the method for producing nicotinamide by a microbial catalysis method has the advantages of high selectivity, high efficiency, mild reaction conditions, less impurities, less environmental pollution and the like. The microbial catalysis reaction is often required to be carried out in a buffer salt system so as to maintain the stability of enzyme activity. Some researches have been focused on the expression of enzyme genes by using escherichia coli or bacillus subtilis as a host, which often needs to be realized by adding an inducer or an antibiotic during the culture of microorganisms, and has high culture cost and poor genetic stability of strains. Therefore, for the microbiological method, a strain with low culture cost, high enzyme activity, strong catalytic ability and good genetic stability is selected, and plays a vital role in the production of the whole nicotinamide.

Disclosure of Invention

The invention aims to provide a Rhodococcus ruber strain and a method for producing nicotinamide by using the Rhodococcus ruber strain so as to solve the problems.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for producing nicotinamide by using Rhodococcus ruber comprises the following process steps:

(1) preparation of a rhodococcus ruber catalyst:

seed culture: adding a culture medium which is 40-90% of the volume of the seeding tank into the seeding tank, wherein the culture medium comprises the following components in percentage by mass: 0.2-1% of yeast extract, 0.5-2% of glucose, 0.1-2% of sodium chloride, 0.02-1% of magnesium sulfate heptahydrate, 0.02-1% of potassium dihydrogen phosphate, 0.02-1% of dipotassium hydrogen phosphate and 0.01-0.5% of defoaming agent; adjusting pH to 6.5-8.0 with sodium hydroxide; sterilizing at 125 ℃ for 25-35 minutes at 118-;

fermenting strains: adding a culture medium which is 40-90% of the volume of the fermentation tank into the fermentation tank, wherein the culture medium comprises the following components in percentage by mass: 0.2-1% of yeast extract, 0.5-2% of glucose, 0.1-5% of corn steep liquor, 0.1-2% of ammonium sulfate, 0.02-1% of magnesium sulfate heptahydrate, 0.02-1% of potassium dihydrogen phosphate, 0.02-1% of dipotassium hydrogen phosphate and 0.01-0.5% of defoaming agent; adjusting pH to 6.5-8.0 with sodium hydroxide; sterilizing at 125 ℃ for 25-35 minutes at 118-;

(2) hydration reaction:

removing culture medium components from fermentation broth after fermentation culture, adding the fermentation broth into a reaction kettle, diluting thalli by pure water, controlling the liquid loading amount to be 60% of the volume of the reaction kettle, controlling the reaction temperature to be 20-60 ℃, slowly adding 3-cyanopyridine in a flowing manner for reaction, stopping adding the 3-cyanopyridine in the flowing manner when the reaction concentration reaches above 400 g/L, tracking the residual amount of the 3-cyanopyridine by liquid chromatography to be less than 0.01, and finishing the reaction to obtain the nicotinamide.

The strain used in the step (1) is Rhodococcus ruber; the Rhodococcus ruber is classified and named Latin Rhodococcus ruber, is preserved in China general microbiological culture Collection center, and has the preservation address: west road No.1 hospital No. 3, north jing, chaoyang district, preservation date: 28/10/2019, accession number: CGMCC No. 18759.

The Rhodococcus ruber cell is in a short rod shape, the bacterial colony is round, orange yellow, gram positive, catalase positive and oxidase negative, and glucose, fructose, sorbitol, citric acid and the like can be utilized. The results of physicochemical tests and 16SrRNA gene sequence determination results are from detection and identification reports of the institute of microbiology of Chinese academy of sciences, and the detailed information is as follows:

1. results of cell morphology and physicochemical experiments

2. 16S rRNA Gene sequence determination results

The method takes nitrile hydratase generated by rhodococcus ruber fermentation as a catalyst, takes pure water as a reaction system, and catalytically converts the 3-cyanopyridine into the nicotinamide by a fed-batch or batch adding mode.

Compared with the prior art, the Rhodococcus ruber for producing nitrile hydratase provided by the invention has the following beneficial effects that the Rhodococcus ruber for producing nitrile hydratase has good genetic stability, low culture cost and high activity of the produced nitrile hydratase, and can be directly used for converting 3-cyanopyridine into nicotinamide, and during catalytic reaction, pure water is used as a reaction system, the final reaction concentration of a reaction liquid can reach more than 400 g/L, so that foreign impurities are avoided, extraction and separation operations are facilitated, and the Rhodococcus ruber has advantages in industrial application.

Detailed Description

The invention is further illustrated by the following specific examples.