CN103146785B - Production process for producing antiviral medicament ribavirin through bacillus amyloliquefaciens precursor addition fermentation method - Google Patents
Production process for producing antiviral medicament ribavirin through bacillus amyloliquefaciens precursor addition fermentation method Download PDFInfo
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- CN103146785B CN103146785B CN201310097836.3A CN201310097836A CN103146785B CN 103146785 B CN103146785 B CN 103146785B CN 201310097836 A CN201310097836 A CN 201310097836A CN 103146785 B CN103146785 B CN 103146785B
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Abstract
The invention discloses a production process for producing antiviral medicament ribavirin through a bacillus amyloliquefaciens precursor TCA addition (1, 2, 4-triazole-3-carboxyformamide) fermentation method. The process comprises the following steps: establishing recombinant plasmid pBSA43-Bs816PNP which can be stably replicated in bacillus amyloliquefaciens cells and efficiently secretes and expresses purine nucleoside phosphorylase (PNPase); converting the plasmid into vernine for producing the bacillus amyloliquefaciens TA208, thereby obtaining a genetically engineered bacterium RBVM (pBSA43-Bs816PNP) with the two characteristics of high-efficiency production and high-efficiency expression of PNPase. By adoption of the genetically engineered bacterium, by taking glucose, yeast extract, bean concentrate, corn steep liquor, monopotassium phosphate, magnesium sulfate, ferrous sulfate and running water as raw materials, the precursor TCA is added for fermenting, and high-concentration ribavirin can be detected in the culture medium after the precursor is added within several hours. The process has the advantages of short fermentation period, high yield, high glucoside conversion rate, easy and convenient process, low cost, low energy consumption, slight pollution and the like.
Description
Technical field
The invention belongs to biochemical engineering field, relate to a kind of technique of genetic engineering bacterium interpolation precursor fermentative Production antiviral drug.
Background technology
Ribavirin (ribavirin), chemistry 1-β-D-RIBOSE base-1H-1 by name, 2,4-triazole-3-carboxylic acid amides, another name virazole, is a kind of broad-spectrum antiviral medicament, multiple DNA and RNA viruses are all had to remarkable restraining effect, in antiviral clinical practice, be used widely.
Ribavirin production method has chemical synthesis, fermentation method and enzyme process three classes.At present, the domestic chemical synthesis of implementing in industrial production accounts for the overwhelming majority, exists by product many, and productive rate is low, and operation steps is many, seriously polluted, high in cost of production deficiency.
Production by Enzymes ribavirin is initiated by Witkowski etc., grow up in eighties of last century 80, the nineties, be widely used in subsequently the production of ribavirin, but the enzyme source cell production cost of Production by Enzymes ribavirin is higher, must first after being separated, thalline just can put in reaction, yeast culture process and ribavirin production process are divided into two stages, can not realize coupling, and need to maintain higher temperature of reaction.
It is low that interpolation precursor fermentation method (Japanese Laid-Open Patent 17830/1979) has raw materials cost, wide material sources, energy consumption is low, the process that yeast culture and ribavirin are produced is synchronizeed and the advantage such as is carried out, but up to the present, this method output is lower, still in the laboratory study stage, can not put in the middle of industrial production, its key reason is: due to technical bottleneck, current ribavirin is produced bacterial strain can not have High-efficient Production nucleosides and these two kinds of features of high efficient expression purine nucleoside phosphorylase concurrently.
Summary of the invention
The object of the invention is in order to solve the above-mentioned problems in the prior art, a kind of production technique of utilizing bacillus amyloliquefaciens to add precursor fermentative Production antiviral drug is provided.
This technique is produced fungi degradation bacillus amyloliquefaciens TA208 as transformation object take guanosine, increase substantially the ability of this bacterium purine biosynthesis nucleoside phosphorylase (PNPase), break through the bottleneck that all the time can not break through, realize as long as add precursor TCA(1 in substratum, 2,4-triazole-3-carboxylic methane amide) just can direct production ribavirin.Its advantage is that technique is simple, and with short production cycle, output is high, and glucosides transformation efficiency is high, does not need hot conditions, has reduced the production cost of ribavirin.
Bacillus amyloliquefaciens provided by the invention adds the production technique of precursor fermentative Production antiviral drug, and its step is as follows:
A, utilize Protocols in Molecular Biology to produce fungi degradation bacillus amyloliquefaciens TA208 using guanosine and build the genetic engineering bacterium RBVM(pBSA43-Bs816PNP that has high efficient expression purine nucleoside phosphorylase (PNPase) concurrently as Host Strains): utilize the round pcr lower molecular weight purine nucleoside phosphorylase encoding gene pupG that increases from subtilis BS168, be connected with the expression vector pBSA43 of process transformation, build and had compared with the secreted expression carrier pBSA43-Bs816PNP of high expression level efficiency and very high plasmid stability and engineering bacteria RBVM(pBSA43-Bs816PNP), detailed process is shown in embodiment 1.
(note: guanosine is produced the seed selection of fungi degradation bacillus amyloliquefaciens TA208 and the research of zymotechnique is completed in 2005 by the Wu Gaihong in this laboratory; Clonal expression, recombinant protein purification and the zymologic property research of the purine nucleoside phosphorylase gene of coding high enzymatic activity are delivered in " Chinese biological engineering magazine " the 30th the 12nd phase of volume in December, 2010 by the Xia Jungang in this laboratory.Through the ribavirin engineering bacteria RBVM(pBSA43-Bs816PNP that further transformation obtains) be now preserved in metabolic engineering research department of University Of Science and Technology Of Tianjin.）
B, engineering bacteria adds precursor fermentation: by the bacterial classification RBVM(pBSA43-Bs816PNP through enlarged culturing obtaining in steps A) access fermention medium carries out engineering bacteria and adds precursor fermentation, in fermention medium, carbon source content should be 8%～12%, carbon-nitrogen ratio N:C should be between 10:100～15:100, inorganic salt content should be 0.1%～12%, initial pH7.2～7.8 of fermention medium, 33 ℃～35 ℃ of culture temperature, shaking culture or fermentor cultivation 60～80h, inoculum size is 5%～10%(V/V), in fermenting process, adding ammoniacal liquor regulates the pH value of fermented liquid to maintain 7.0～7.4, fermentation starts 12-24h and adds TCA.
(1). fermention medium
Glucose 8%～12%, yeast extract paste 0.8%～1.0%, beans are dense 1.6%～2%, corn steep liquor 1%～2%, (NH
3)
2sO
41%～1.5%, MgSO
47H
2o 0.1%～0.15%, KH
2pO
40.1%～0.3%, FeSO
42～4mg/L, tap water 96%～98%, Medium's PH Value is 7.0～7.4.
(2). fermentating controling process
Adopt 5L fermentor tank, 33～35 ℃ of leavening temperatures, air flow quantity is 0.8～8.0L/min, tank pressure 0.001～0.006MPa, dissolved oxygen maintains 15～40%, and fermentation pH value is controlled at 7.0～7.4, and fermentation starts 12-24h interpolation TCA, continues to ferment to 60-80h and finishes.
C, the quantitative analysis of product ribavirin:
Sample centrifugally, get supernatant liquor and dilute suitable multiple through aseptic deionized water, adopt high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) detection by quantitative ribavirin.Working conditions is: Agilent 1200 type high performance liquid chromatographs; Detector VWD(Variable wavelength UV detector); Detect wavelength 207nm; Sampler standard type automatic sampler; Sample size 5 μ L; Chromatographic column Kromasil C18,5 μ, 250mm × 4.6mm; Moving phase water: acetonitrile=96:4(V/V); 30 ℃ of column temperatures; Flow velocity 1ml/min.
The definition of ribavirin transformation efficiency:
Advantage of the present invention and positively effect
1) adopt genetic engineering means, broken through guanosine high yield and the high efficient expression of PNPase cannot be on same strain bacterium accomplished bottleneck, make PNPase be able to produce in fungi degradation bacillus amyloliquefaciens TA208 and obtain high efficient expression at guanosine, and can well be secreted in substratum and keep active, the PNPase vigor before transformation is improved largely.
2) simple to operate, adopt traditional zymotechnique, equipment that the present invention is easily realized universal, leavening temperature is controlled at 33～35 ℃, avoids consuming the extra energy in order to maintain compared with high invert point.
3) fermentation period is short, and output is high, higher Content of Ribavirin can in fermented liquid, be detected through fermentation in 60-80 hour, reaches as high as at present 27g/L, glucosides transformation efficiency 35%-44%.
Accompanying drawing explanation
Fig. 1 is that transformant extracts plasmid enzyme restriction checking.
Embodiment
Embodiment 1 builds genetic engineering bacterium RBVM(pBSA43-Bs816PNP)
Adopt associated molecule biology techniques to transform rear called after pBSA43 to carrier pBE2, it has following features: be 1. equipped with the P43 promotor that can be identified by bacillus amyloliquefaciens RNA polymerase, guarantee goal gene can be in Host Strains high efficient expression, and without chemical inducer IPTG or lactose-induced.2. for the feature of bacillus amyloliquefaciens restriction modification system, removed the BamH I restriction enzyme site on plasmid, plasmid is more easily transformed; 5 ' the end that 3. sequence of coded signal peptide in sacB gene order can be fused to goal gene, can be secreted in substratum goal gene.
The structure of pBSA43 plasmid: adopt round pcr, respectively by the sequence of coded signal peptide in the sequence of P43 promotor and sacB gene order clonal expansion from subtilis BS168 genome, be inserted into the multiple clone site of pBE2 plasmid, make the 5 ' downstream of holding 3 ' end of the sequence that is positioned at P43 promotor of the sequence of coded signal peptide in sacB gene order, the novel plasmid called after pBSA43 of formation.The primer that the pcr amplification of P43 promotor adopts is referring to sequence table SEQ ID No.1 and SEQ ID No.2, and sequence is shown in sequence table SEQ ID No.7; The primer that the pcr amplification of SacB signal peptide adopts is referring to sequence table SEQ ID No.3 and SEQ ID No.4, and sequence is referring to sequence table SEQ ID No.8.
The structure of recombinant plasmid pBSA43-Bs816PNP: adopt round pcr, by gene pupG clonal expansion from subtilis BS168 genome of coding purine nucleoside phosphorylase, and recombinate with pBSA43 plasmid, make 5 ' of pupG sequence hold the downstream of 3 ' end of the sequence that is arranged in sacB gene order coded signal peptide (noting guaranteeing the complete of reading frame herein), built the recombinant plasmid pBSA43-Bs816PNP of high efficient expression purine nucleoside phosphorylase.The primer that the pcr amplification of pupG adopts is referring to sequence table SEQ ID No.5 and SEQ ID No.6, and sequence is referring to sequence table SEQ ID No.9.
The conversion of recombinant plasmid: according to the feature of bacillus amyloliquefaciens tool restriction modification system, avoid selecting BamH I as restriction enzyme site building in plasmid as far as possible, and guarantee not contain on plasmid BamH I restriction enzyme site as far as possible.By electric shock conversion method, recombinant plasmid pBSA43-Bs816PNP is transformed to guanosine and produce fungi degradation bacillus amyloliquefaciens TA208, getting 1～10 μ L concentration and be 100ng/ μ L recombinant plasmid solution joins in 100 μ L competent cells and is transferred in the electric revolving cup of 2mm ice bath precooling after ice bath 30min, the conversion of shocking by electricity under 1800V voltage, after electric shock, add immediately 900 μ L LB substratum, 35 ℃ of shaking culture 2h, rotating speed is 220r/min, the cultivation of recovering.Recovery is got 200 μ L nutrient solutions and is applied to containing on the LB flat board of 10 μ g/L kantlex after cultivating and finishing, be inverted for 35 ℃ and cultivate 18h, after growing bacterium colony, picking list colony inoculation is in the 35 ℃ of shaking culture 12h in pipe that shake that 5ml LB substratum is housed, rotating speed is 220r/min, upgrading grain carries out enzyme and cuts checking, the results are shown in Figure 1.
Embodiment 2 engineering bacteria RBVM(pBSA43-Bs816PNP) plasmid stability
Adopt the dull and stereotyped dilution method of counting.Frozen engineering bacteria (embodiment 1 builds) in-80 ℃ of glycerine pipes, containing three rides on the LB flat board of 10 μ g/L kantlex, is cultivated to 16h for 37 ℃, and picking list colony inoculation is in 5ml fitting of fluids substratum (LB/Km
r) in, shake after pipe is cultivated 12h and proceed to 5ml liquid Nonsele ctive culture media (LB) with 1% inoculum size, start to shake continuously pipe culture experiment.Once, inoculum size is 1% to every 12h transferred species, and continuous passage 30 times is carried out the dull and stereotyped disappearance degree that detects plasmid of diluting every 5 sub-samplings.When detection, get bacterium liquid lml sterilized water stepwise dilution, get three suitable gradients, this experimental selection 10
-7, 10
-8, 10
-9three gradients are coated in respectively on solid selective medium and Nonsele ctive culture media flat board, three repetitions of each processing.Be upside down in 37 ℃ of incubators and cultivate 16h, calculate the colony number in each ware.Colony number in colony number in selective medium and Nonsele ctive culture media is compared, can calculate according to formula the miss rate of plasmid, in table 1.
Table 1 engineering bacteria RBVM(pBSA43-Bs816PNP) plasmid structural stability
Plasmid loss rate calculation formula:
Embodiment 3 engineering bacteria RBVM(pBSA43-Bs816PNP) fermentation of shaking flask product ribavirin
From fresh activated inclined plane respectively picking one encircle the engineering bacteria RBVM(pBSA43-Bs816PNP that embodiment 1 builds) lawn (glucose 2%, extractum carnis 1%, (NH in seed culture medium
3)
2sO
41%, yeast powder 0.75%, MgSO
47H
2o 0.1%, KH
2pO
40.1%, corn steep liquor 0.2%, beans are dense 0.2%, pH7.0～7.2), 500mL triangular flask liquid amount 25mL, 9 layers of gauzes sealing.Be placed in 35 ℃ of shaking culture 10h on rotary shaking table, rotating speed is 220r/min.
So that in 5% inoculum size access fermention medium, (beans are dense 1.6%, corn steep liquor 1%, (NH for glucose 8%, yeast extract paste 0.8%
3)
2sO
41%, MgSO
47H
2o 0.1%, KH
2pO
40.1%, FeSO
42mg/L, pH7.3～7.4).500mL triangular flask liquid amount 30mL, 33 ℃ of shaking culture 60h, rotating speed is 220r/min.Regulate pH value in 7.0～7.2 with 25% ammoniacal liquor, maintain residual sugar in 1%～3% with 80% glucose.Fermentation starts 24h and adds precursor TCA15g/L, ferments to 60h and finishes.Cell density OD
600reach as high as 24, ribavirin output reaches as high as 12.5g/L.
Embodiment 4 engineering bacteria RBVM(pBSA43-Bs816PNP) fermentation of shaking flask product ribavirin
Inclined-plane seed activation cultivation, seed culture medium and fermentation are controlled as embodiment 3.Access in fermention medium with inoculum size 8% that (beans are dense 1.8%, corn steep liquor 1.5%, (NH for glucose 10%, yeast extract paste 0.9%
3)
2sO
41.2%, MgSO
47H
2o 0.12%, KH
2pO
40.2%, FeSO
43mg/L, pH7.2～7.4).The bottled liquid measure 30mL of 500mL baffle plate, 34 ℃ of shaking culture 70h, rotating speed is 220r/min.Regulate pH value in 7.0～7.2 with 25% ammoniacal liquor, maintain residual sugar in 2%～4% with 80% glucose.Fermentation starts 18h and adds precursor TCA 15g/L, ferments to 70h and finishes.Cell density OD
600reach as high as 27, ribavirin output reaches as high as 14g/L.
Embodiment 5 engineering bacteria RBVM(pBSA43-Bs816PNP) fermentation of shaking flask product ribavirin
Inclined-plane seed activation cultivation, seed culture medium and fermentation are controlled as embodiment 3.Access in fermention medium with inoculum size 10% that (beans are dense 2%, corn steep liquor 2%, (NH for glucose 12%, yeast extract paste 1.0%
3)
2sO
41.5%, MgSO
47H
2o 0.15%, KH
2pO
40.3%, FeSO
44mg/L, pH7.0～7.2).The bottled liquid measure 30mL of 500mL baffle plate, 35 ℃ of shaking culture 80h, rotating speed is 220r/min.Regulate pH value in 7.0～7.2 with 25% ammoniacal liquor, maintain residual sugar in 4%～6% with 80% glucose.Fermentation starts 12h and adds precursor TCA 15g/L, ferments to 80h and finishes.Cell density OD
600reach as high as 28, ribavirin output reaches as high as 20g/L.
Embodiment 6 engineering bacteria RBVM(pBSA43-Bs816PNP) fermentation of 5L tank product ribavirin
From fresh activated inclined plane respectively picking one encircle engineering bacteria RBVM(pBSA43-Bs816PNP) lawn is in seed culture medium, inclined-plane seed activation cultivation, seed and fermention medium are with embodiment 3,33 ℃ of leavening temperatures, tank pressure 0.001～0.006MPa, air flow quantity 0.8～8.0L/min and stirring velocity 200～700r/min make dissolved oxygen maintain 15～40%, fermentation pH value is controlled between 7.0～7.2, and fermentation starts 12～24h interpolation precursor TCA 15g/L, ferments to 80h and finishes.Cell density OD
600reach as high as 33, ribavirin output reaches as high as 24g/L.
Embodiment 7 engineering bacteria RBVM(pBSA43-Bs816PNP) fermentation of 5L tank product ribavirin
From fresh activated inclined plane respectively picking one encircle engineering bacteria RBVM(pBSA43-Bs816PNP) lawn is in seed culture medium, inclined-plane seed activation cultivation, seed and fermention medium are with embodiment 5,35 ℃ of leavening temperatures, tank pressure 0.001～0.006MPa, air flow quantity 0.8～8.0L/min and stirring velocity 200～700r/min make dissolved oxygen maintain 15～40%, fermentation pH value is controlled between 7.0～7.2, and fermentation starts 12～24h interpolation precursor TCA 15g/L, ferments to 80h and finishes.Cell density OD
600reach as high as 36, ribavirin output reaches as high as 27g/L.
Claims (3)
1. a bacillus amyloliquefaciens adds precursor TCA(1,2,4-triazole-3-carboxylic methane amide) production technique of fermentative Production antiviral drug, utilize the genetic engineering bacterium RBVM(pBSA43-Bs816PNP that has High-efficient Production nucleosides and high these two kinds of features of efficient expression purine nucleoside phosphorylase concurrently building voluntarily) realize fermentative Production ribavirin by adding precursor TCA in fermenting process, concrete technology comprises:
A. utilize Protocols in Molecular Biology to produce fungi degradation bacillus amyloliquefaciens TA208 using guanosine and build the genetic engineering bacterium RBVM(pBSA43-Bs816PNP that has high efficient expression purine nucleoside phosphorylase (PNPase) concurrently as Host Strains): utilize the round pcr lower molecular weight purine nucleoside phosphorylase encoding gene pupG that increases from subtilis BS168, be connected with the expression vector pBSA43 of process transformation, build and had compared with the secreted expression carrier pBSA43-Bs816PNP of high expression level efficiency and very high plasmid stability and engineering bacteria RBVM(pBSA43-Bs816PNP),
B. engineering bacteria adds precursor fermentation: by the bacterial classification RBVM(pBSA43-Bs816PNP through enlarged culturing obtaining in steps A) access fermention medium carries out engineering bacteria and adds precursor fermentation, in fermention medium, carbon source content should be 8%～12%, carbon-nitrogen ratio N:C should be between 10:100～15:100, inorganic salt content should be 0.1%～12%, initial pH7.2～7.8 of fermention medium, 33 ℃～35 ℃ of culture temperature, shaking culture or fermentor cultivation 60～80h, inoculum size is 5%～10%V/V, in fermenting process, adding ammoniacal liquor regulates the pH value of fermented liquid to maintain 7.0～7.4, fermentation starts 12-24h and adds TCA.
2. production technique according to claim 1, is characterized in that B step produces the substratum that ribavirin fermentation adopts: glucose 8%～12%, and yeast extract paste 0.8%～1.0%, beans are dense 1.6%～2%, corn steep liquor 1%～2%, (NH
3)
2sO
41%～1.5%, MgSO
47H
2o 0.1%～0.15%, KH
2pO
40.1%～0.3%, FeSO
42～4mg/L, tap water 96%～98%, fermentation pH value is controlled at 7.0～7.4, and fermentation starts 12-24h and adds TCA.
3. production technique according to claim 1, is characterized in that the control technique that the fermentation of B step production ribavirin adopts: 33～35 ℃ of leavening temperatures, and dissolved oxygen maintains 15～40%, and fermentation time is 60～80h.
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