CN104762360A - High-content nicotinamide synthesis induced by new-feature nitrile hydratase - Google Patents
High-content nicotinamide synthesis induced by new-feature nitrile hydratase Download PDFInfo
- Publication number
- CN104762360A CN104762360A CN201510115659.6A CN201510115659A CN104762360A CN 104762360 A CN104762360 A CN 104762360A CN 201510115659 A CN201510115659 A CN 201510115659A CN 104762360 A CN104762360 A CN 104762360A
- Authority
- CN
- China
- Prior art keywords
- nitrile hydratase
- niacinamide
- reaction
- rhodococcus
- content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method of producing nicotinamide through microorganism catalysis. According to a synergistic effect theory, a novel binary composite nitrile hydratase is disclosed, wherein the composite nitrile hydratase is produced through combined cultivation of bacillus pallidus and rhodococcus. The microorganism nitrile hydratase, which is characterized by being high in stereoselectivity, regioselectivity and being resistant to substrates and products in high concentration, is obtained by means of high substrate specificity and thermostability of the bacillus pallidus and a strong promoter effect of the rhodococcus. Meanwhile, by means of introduction of a co-solvent at a certain concentration to the reaction system, the reaction is accelerated. The method is simple and high-efficient, is environment-friendly, can reach not less than 99% in both purity and yield of the product and is suitable for large-scale industrial production.
Description
Technical field
The invention belongs to microbial technology field.Be specifically related to a kind of method that new features Nitrile hydratase catalysis method produces niacinamide.
Background technology
Niacinamide, also known as nicotinamide, belongs to B race microorganism, is second largest VITAMIN in the 14 kinds of VITAMIN in the whole world, is only second to vitamin-E in current usage quantity, and whole world year usage quantity reaches 60000 tons at present.Niacinamide is mainly present in animal body, clinical treatment mostly be synthetics.Lack eupnea and metabolism that niacinamide can affect cell, thus produce pellagra, stomatitis, glossitis, hepatic diseases and sunburn.Heavy dose of niacinamide can reduce the amount of cholesterol and triglyceride level, its physiological action and hydrochloric acid similar, but better water-soluble, without obvious vasodilation effect.Be widely used in the industries such as medicine, food, feed, makeup, chemical industry.
Current niacinamide production technique has chemical method and microbial method.What chemical method adopted is that 5-pentamethylene diamine, quinoline etc. are raw material with 3-picoline, 2-methyl isophthalic acid, generates nicotinonitrile by oxygenant direct oxidation or catalyzed oxidation, then produces niacinamide through alkali lye hydrolysis.This technique is more ripe, but need add catalyzer, high temperature, polystep reaction, and after reaction, the reaction solution content of gained is not high, and component is impure, is unfavorable for extracting.Biological catalysis adopts the Nitrile hydratase produced in microorganism cells to transform nicotinonitrile to generate niacinamide.Microbial method is produced niacinamide and is had reaction conditions gentleness, and flow process is simple, products collection efficiency and purity advantages of higher, these be all chemical synthesis incomparable.Thus microbial method receives the concern of investigator day by day.
United States Patent (USP) (USA4008241) adopts chemical method, prepares niacinamide with nicotinonitrile under ammonia soln effect.This temperature of reaction 90-150 DEG C, reaction times 4-8 h, the maximum conversion rate of its nicotinonitrile only 70%.And by multistep sepn process, unconverted nicotinonitrile need be separated in product nicotinamide with ammonia, cost is high, and product is impure; United States Patent (USP) (USA1133013) adopts chemical method equally, carries out the catalytic hydration reaction of nitrile, gained niacinamide yield only 79.28% with Manganse Dioxide, and has the shortcomings such as material toxicity is large, the required catalytic amount of reaction is high.
Chinese patent (CN1424402A) reports a kind of method of microorganism catalytic processes for niacinamide production, it adopts the Nitrile hydratase of propionic acid bar bacterium that nicotinonitrile is changed into niacinamide, avoid and make chemical method catalyzer usage quantity large and the feature that reactions steps is many, but the preparation of zymogenic cells is comparatively loaded down with trivial details; It is substrate that Chinese patent (CN101712944B) reports with nicotinonitrile, take subtilis as biological catalyst, the mode adopting substrate and biological catalyst to be coupled to add is carried out catalytic hydration and is reacted to obtain niacinamide, and the purity of gained niacinamide is not high, only about 98%.
The present inventor is by a large amount of experiment screenings and optimization, determine suitable microbe species and catalysis process thereof, overcome the problems referred to above, the purity of niacinamide and yield are improved greatly, and the aftertreatment purity of the niacinamide simple and regular obtained can reach more than 99.9%.
Summary of the invention
In order to make up shortcomings and deficiencies of the prior art, the object of the present invention is to provide a kind of production method of niacinamide, employing has excellent synergistic binary compound Nitrile hydratase, introduces certain density solubility promoter, effectively raise purity and the yield of product in simultaneous reactions system.Synthetic method is simple to operate, efficient, environmental friendliness, loss are little.
The technical solution used in the present invention is as follows to achieve these goals:
The method of the high-content niacinamide synthesis of new features Nitrile hydratase induction, is characterized in that: employing be binary compound Nitrile hydratase, this prozyme produces by being combined to cultivate by genus bacillus and rhodococcus in vain, concrete synthetic method:
White being inoculated into by genus bacillus and rhodococcus of being preserved by glycerine pipe is applicable to whitely be carried out fermentation culture by the substratum of genus bacillus and rhodococcus, then gauze cleaning is carried out to fermented liquid, obtain thalline, somatic cells or the somatic cells that processed are added to containing the reaction that is hydrolyzed in nicotinonitrile solution, finally conversion fluid is separated, obtains niacinamide.
The high-content niacinamide synthetic method of described new features Nitrile hydratase induction, is characterized in that, comprise the following steps:
(1) seed culture: seed culture medium forms: 0.5 ~ 8% glycerine, 0.2 ~ 8% yeast extract paste, 0.05 ~ 0.5%NaCl, 0.05 ~ 0.5% K
2hPO
4, 0.05 ~ 0.5% MgSO
4, 0.1 ~ 1% urea, with deionized water preparation, pH is moderate; Get seed culture medium, in packing triangular flask, sterilizing, then accesses bacterial classification in vain by genus bacillus and rhodococcus, shaking speed 80 ~ 450 r/min, cultivates 25 ~ 85h as seed liquor at 15 ~ 50 DEG C of shaking tables, for subsequent use;
(2) fermentation culture: fermention medium forms: 1 ~ 8% glycerine, the glucose of 0.1 ~ 5% or sucrose, 0.2 ~ 3% yeast powder, the urea of 0.1 ~ 5%, 0.05 ~ 0.8% K
2hPO
4, 0.05 ~ 0.8% MgSO
4, 1 ~ 30ppm CoCl
2, with deionized water preparation, by fermention medium packing, sterilizing, access seed liquor, pH 6.5 ~ 8.5, cultivates 10 ~ 25 h at temperature 20 ~ 80 DEG C;
(3) catalytic hydrolysis: fermentation liquor gauze is cleaned, obtains pure nitrile hydratase production parent cell, nitrile hydratase production cell and deionized water are suspended in deionized water according to the mass ratio of 1:10, then join in reactor in batches, the scale dimension of enchylema and reaction solution volume is held in >=and 1%, solubility promoter also containing volumetric concentration 0.1-15% in reaction solution, at the temperature of 5 ~ 85 DEG C, enzymic catalytic reaction is carried out in stirring, substrate and catalyzer is adopted to be coupled additional way in batches in reaction process, solid or the solution of adding nicotinonitrile make concentration of substrate control 20 ~ 60%, through the reaction of 6 ~ 48h, obtain the nicotinamide soln of concentration more than 20%, concentration accumulation >=20% after disposable go out tank, certain density reaction solution will be reached and remove cell and cell debris by micro-filtration, obtain nicotinamide soln,
(4) purification process: reaction solution is removed protein through nanofiltration, then adds activated carbon decolorizing, then concentrates, and the solution after concentrated carries out mist projection granulating again and air stream drying must arrive the niacinamide of finished product purity more than 99.9%.
The high-content niacinamide synthetic method of described new features Nitrile hydratase induction, is characterized in that: described solubility promoter is selected from: the one in methyl alcohol, ethanol, methyl-sulphoxide, ethylene glycol, PEG-4000, PEG-8 00, chloroform, acetone.
Add glucose or the sucrose of 0.1 ~ 5% in fermention medium composition, the Rapid Accumulation of Fungal biodiversity can be promoted, shorten the lag period in thalline fermentation production process, reduce thalli growth to the consumption of substrate glycerol.
The advantage of the high-content niacinamide synthetic method of binary compound Nitrile hydratase induction provided by the invention:
1) prior art is used for the bacterium of bacterial strain that catalysis nitrile becomes acid amides mainly Rhod, and the binary compound Nitrile hydratase of a kind of novelty that what the present invention adopted is, this prozyme produces by being combined to cultivate by genus bacillus and rhodococcus in vain.This compound Nitrile hydratase has substrate and the product of highly-solid selectively, regioselectivity and resisting high-concentration, and total enzyme activity of unit volume fermented liquid is up to 1500U.
2) adding renewable carbohydrate in fermention medium of the present invention is auxiliary carbon source, make full use of renewable carbohydrate resource on the one hand, the Rapid Accumulation of Fungal biodiversity can be promoted on the other hand, shorten the lag period in thalline fermentation production process, reduce thalli growth to the consumption of substrate glycerol; Fermentation period shortens 30 ~ 50 h than congenic method, and growth pressure reaches 4 ~ 6 g/Lh.
3) the present invention adds the solubility promoter of volumetric concentration 0.1-15%, adopts substrate and catalyzer coupling to add in batches, effectively reduces high concentration substrate, the toxic action of products upon cell and biological catalyst downtrod cumulative time.
With biological process synthesis niacinamide, adopt and have excellent synergistic binary compound Nitrile hydratase, the purity of niacinamide and yield all can reach more than 99.9%.Synthetic method is simple and reliable, convenient operation, production cost are low, environment improves.
Embodiment
A method for the high-content niacinamide synthesis of new features Nitrile hydratase induction, employing be binary compound Nitrile hydratase, this prozyme produces by being combined to cultivate by genus bacillus and rhodococcus in vain, and concrete synthetic method, comprises the following steps:
(1) at 1m
3triangular flask or fermentor tank in load 50% seed culture medium, after sterilizing access 20% bacterial classification, at 30 DEG C in rotary shaker shaking culture 35 h, then fermentor tank load 50% substratum, the seed liquor of access 6% after real tank sterilization, at air flow 1:08(V. V. M); Tank pressure 0.06MPa; Temperature: 35 DEG C, ferment 15 h;
Seed culture medium forms: 2% glycerine, 1% yeast extract paste, 0.3%NaCl, 0.2% K
2hPO
4, 0.1% MgSO
4, 0.05% urea, pH7.0 ~ 8.5,
Fermention medium forms: 2% glycerine, 0.5% glucose, 1.2% yeast powder, the urea of 2%, 0.15% K
2hPO
4, 0.5% MgSO
4, 150ppm CoCl
2, pH6.5 ~ 8.5.
(2) by 100mL fermented liquid through cleaning microfiltration membranes, obtain the pure nitrile hydratase production parent cell aqueous solution; By cell suspension in the deionized water containing 10ml ethanol, the scale dimension of enchylema and reaction solution volume is held in 3%, at the temperature of 35 DEG C, stirs and carry out enzymic catalytic reaction under 150rpm rotating speed.Solid or solution by adding nicotinonitrile in reaction process make concentration of substrate control 30%, and through the reaction of 25h, the nicotinonitrile of total 20Kg is converted into niacinamide, transformation efficiency 99.9%.
(3) nicotinamide soln that hydration reaction obtains is removed protein through nanofiltration, then add activated carbon decolorizing, then concentrate, the solution after concentrated carries out the tobacco product acid amides that mist projection granulating and air stream drying must arrive purity 99.9% again.
Claims (3)
1. the method for the high-content niacinamide synthesis of new features Nitrile hydratase induction, is characterized in that: employing be binary compound Nitrile hydratase, this prozyme produces by being combined to cultivate by genus bacillus and rhodococcus in vain, concrete synthetic method:
White being inoculated into by genus bacillus and rhodococcus of being preserved by glycerine pipe is applicable to whitely be carried out fermentation culture by the substratum of genus bacillus and rhodococcus, then gauze cleaning is carried out to fermented liquid, obtain thalline, somatic cells or the somatic cells that processed are added to containing the reaction that is hydrolyzed in nicotinonitrile solution, finally conversion fluid is separated, obtains niacinamide.
2. the high-content niacinamide synthetic method of new features Nitrile hydratase induction according to claim 1, is characterized in that, comprise the following steps:
(1) seed culture: seed culture medium forms: 0.5 ~ 8% glycerine, 0.2 ~ 8% yeast extract paste, 0.05 ~ 0.5%NaCl, 0.05 ~ 0.5% K
2hPO
4, 0.05 ~ 0.5% MgSO
4, 0.1 ~ 1% urea, with deionized water preparation, pH is moderate; Get seed culture medium, in packing triangular flask, sterilizing, then accesses bacterial classification in vain by genus bacillus and rhodococcus, shaking speed 80 ~ 450 r/min, cultivates 25 ~ 85h as seed liquor at 15 ~ 50 DEG C of shaking tables, for subsequent use;
(2) fermentation culture: fermention medium forms: 1 ~ 8% glycerine, the glucose of 0.1 ~ 5% or sucrose, 0.2 ~ 3% yeast powder, the urea of 0.1 ~ 5%, 0.05 ~ 0.8% K
2hPO
4, 0.05 ~ 0.8% MgSO
4, 1 ~ 30ppm CoCl
2, with deionized water preparation, by fermention medium packing, sterilizing, access seed liquor, pH 6.5 ~ 8.5, cultivates 10 ~ 25 h at temperature 20 ~ 80 DEG C;
(3) catalytic hydrolysis: fermentation liquor gauze is cleaned, obtains pure nitrile hydratase production parent cell, nitrile hydratase production cell and deionized water are suspended in deionized water according to the mass ratio of 1:10, then join in reactor in batches, the scale dimension of enchylema and reaction solution volume is held in >=and 1%, solubility promoter also containing volumetric concentration 0.1-15% in reaction solution, at the temperature of 5 ~ 85 DEG C, enzymic catalytic reaction is carried out in stirring, substrate and catalyzer is adopted to be coupled additional way in batches in reaction process, solid or the solution of adding nicotinonitrile make concentration of substrate control 20 ~ 60%, through the reaction of 6 ~ 48h, obtain the nicotinamide soln of concentration more than 20%, concentration accumulation >=20% after disposable go out tank, certain density reaction solution will be reached and remove cell and cell debris by micro-filtration, obtain nicotinamide soln,
(4) purification process: reaction solution is removed protein through nanofiltration, then adds activated carbon decolorizing, then concentrates, and the solution after concentrated carries out mist projection granulating again and air stream drying must arrive the niacinamide of finished product purity more than 99.9%.
3. the high-content niacinamide synthetic method of new features Nitrile hydratase induction according to claim 2, is characterized in that: described solubility promoter is selected from: the one in methyl alcohol, ethanol, methyl-sulphoxide, ethylene glycol, PEG-4000, PEG-8 00, chloroform, acetone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510115659.6A CN104762360A (en) | 2015-03-17 | 2015-03-17 | High-content nicotinamide synthesis induced by new-feature nitrile hydratase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510115659.6A CN104762360A (en) | 2015-03-17 | 2015-03-17 | High-content nicotinamide synthesis induced by new-feature nitrile hydratase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104762360A true CN104762360A (en) | 2015-07-08 |
Family
ID=53644451
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510115659.6A Pending CN104762360A (en) | 2015-03-17 | 2015-03-17 | High-content nicotinamide synthesis induced by new-feature nitrile hydratase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104762360A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701725A (en) * | 2017-01-19 | 2017-05-24 | 安徽瑞邦生物科技有限公司 | Fermentation medium for improving activity of bacillus subtilis nitrile hydratase and application |
| CN106755166A (en) * | 2016-12-09 | 2017-05-31 | 江南大学 | The method that HMW engineered strain for nitrile hydratase catalyzes and synthesizes niacinamide |
| CN111039861A (en) * | 2019-12-29 | 2020-04-21 | 安徽瑞邦生物科技有限公司 | Nicotinamide synthesis catalysis process containing low-smoke acid by-product |
| CN111424061A (en) * | 2020-04-01 | 2020-07-17 | 山东昆达生物科技有限公司 | Rhodococcus ruber and method for producing nicotinamide by using same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730660A (en) * | 2004-08-05 | 2006-02-08 | 上海市农药研究所 | Microorganism catalytic processes for niacinamide production |
-
2015
- 2015-03-17 CN CN201510115659.6A patent/CN104762360A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730660A (en) * | 2004-08-05 | 2006-02-08 | 上海市农药研究所 | Microorganism catalytic processes for niacinamide production |
Non-Patent Citations (2)
| Title |
|---|
| REBECCA A.等: "Molecular characterisation of a novel thermophilic nitrile hydratase", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| 娄文勇等: "微生物腈水合酶的研究进展", 《分子催化》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755166A (en) * | 2016-12-09 | 2017-05-31 | 江南大学 | The method that HMW engineered strain for nitrile hydratase catalyzes and synthesizes niacinamide |
| CN106755166B (en) * | 2016-12-09 | 2020-03-24 | 江南大学 | Method for catalytically synthesizing nicotinamide by high molecular weight nitrile hydratase engineering bacteria |
| CN106701725A (en) * | 2017-01-19 | 2017-05-24 | 安徽瑞邦生物科技有限公司 | Fermentation medium for improving activity of bacillus subtilis nitrile hydratase and application |
| CN111039861A (en) * | 2019-12-29 | 2020-04-21 | 安徽瑞邦生物科技有限公司 | Nicotinamide synthesis catalysis process containing low-smoke acid by-product |
| CN111424061A (en) * | 2020-04-01 | 2020-07-17 | 山东昆达生物科技有限公司 | Rhodococcus ruber and method for producing nicotinamide by using same |
| CN111424061B (en) * | 2020-04-01 | 2022-07-12 | 山东昆达生物科技有限公司 | Rhodococcus ruber and method for producing nicotinamide by using same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102337233B (en) | Pseudomonas putida and method for producing nicotinic acid or isonicotinic acid through converting Pseudomonas putida | |
| CN107217048A (en) | It is a kind of to catalyze and synthesize aminopeptidase of carnosine and its preparation method and application | |
| CN104762360A (en) | High-content nicotinamide synthesis induced by new-feature nitrile hydratase | |
| CN102367463B (en) | Method for producing glycyrrhetinic acid monoglucuronide through intermittent feed supplement fermentation | |
| CN104805144B (en) | A kind of method of efficiently production L-citrulline | |
| CN104560913A (en) | Method for preparing nuclease P1 by semi-continuous fermentation of penicillium citrinum | |
| CN103333926B (en) | Method for accelerating synthesis of epsilon-polylysine | |
| CN105838751A (en) | Method for improving output of tetramethylpyrazine produced through fermentation by Bacillus | |
| CN105695518A (en) | Method for repeatedly preparing gallic acid by converting tannic acid through immobilized bacteria biological method | |
| CN104762338A (en) | Method of producing nicotinamide by catalysis of rhodococcus | |
| CN104928224B (en) | A kind of ferulic acid production engineering bacterial strain, construction method and bioconversion method | |
| CN101709322B (en) | Method for synthesizing betulic acid by carrying out biocatalysis on betulin | |
| WO2023103543A1 (en) | Method for preparing nuclease p1 | |
| CN104673767A (en) | Method for producing feruloyl esterase | |
| CN104164411A (en) | Method for preparing glucose oxidase | |
| CN107475312B (en) | Method for effectively producing vitamin K2 by improving cell membrane permeability | |
| CN101812437A (en) | Method for producing chymosin by adopting mildew solid-state fermentation | |
| JP7426159B1 (en) | Method for producing carboxylic acid composition | |
| CN104293851A (en) | Method for producing hydroxyethyl pyridine by alternaria alternata | |
| CN109234247A (en) | A kind of glucose oxidase and preparation method thereof | |
| CN104313074A (en) | Method for producing pyridylethanol through penicillium catalysis | |
| CN107400686A (en) | Ellagic acid prepared by a kind of solid state fermentation granatum and preparation method thereof | |
| CN102399845A (en) | VitB12 fermentation production control process based on CO<2> concentration in tail gas | |
| CN104342464A (en) | Method for producing chiral phenyl methanol employing catalysis of tarlaromyces flavus | |
| CN104263776A (en) | Method for producing chiral pyridine ethanol through biological catalysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150708 |