CN100552037C - The biosynthetic means of 5 ' flavour nucleotide - Google Patents
The biosynthetic means of 5 ' flavour nucleotide Download PDFInfo
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- CN100552037C CN100552037C CNB2006101652760A CN200610165276A CN100552037C CN 100552037 C CN100552037 C CN 100552037C CN B2006101652760 A CNB2006101652760 A CN B2006101652760A CN 200610165276 A CN200610165276 A CN 200610165276A CN 100552037 C CN100552037 C CN 100552037C
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Abstract
The invention discloses a kind of biosynthetic means of 5 ' flavour nucleotide.This method is that enteroaerogen (Enterobacter aerogenes) the TAM1183 thalline that will contain acid phosphatase gene adds in the reaction soln that contains substrate tetra-sodium and inosine or guanosine, at 25-35 ℃, pH 4.0-8.4 is reaction down, obtains 5 '-t-inosinic acid or 5 '-guanylic acid.The present invention has the following advantages: the bacterial strain of 1) selecting for use has the activity of acid phosphatase height, and the advantage that fast growth and environmental compatibility are strong all can be grown under aerobic and anaerobic condition, is suitable for suitability for industrialized production; 2) production method is simple, the output height of purpose product; 3) raw material sources are extensive, and are low for equipment requirements, low production cost.The present invention will play a significant role in the suitability for industrialized production of 5 ' flavour nucleotide, have a extensive future.
Description
Technical field
The present invention relates to the preparation method of 5 ' flavour nucleotide, particularly relate to a kind of method of biosynthesizing 5 ' flavour nucleotide.
Background technology
Eighties of last century fifties, the researchist finds that ((5 '-GMP) produces synergistic effect (claiming synergism again) with Sodium Glutamate (MSG) to 5 '-IMP) and 5 '-guanylic acid to 5 '-t-inosinic acid when mixing, can make the food freshness improve several times to tens times.Therefore, be that the application of the flavour nucleotide of representative has caused extensive concern both domestic and external with 5 '-t-inosinic acid and 5 '-guanylic acid.Nowadays, flavour nucleotide has been widely used in fields such as health care, nutrition, food and medicine as a kind of novel additive.
Since the sixties in last century, Japan took the lead in carrying out the suitability for industrialized production of flavour nucleotide, the method for existing multiple production flavour nucleotide and derivative thereof.Conclude by production technique, mainly contain three kinds: chemical synthesis, RNA enzymolysis process and microbe fermentation method.Production technique of these three kinds of methods and products thereof all has very big difference.
Chemical synthesis: mainly be to utilize nucleosides to carry out phosphating reaction.Phosphorylation agent commonly used mainly is the reactive derivative of phosphoric acid or tetra-sodium.The reactive derivative of tetra-sodium can be pyrophosphoryl chloride or two-right-oil of mirbane tetra-sodium etc., and industrial extensive employing is phosphoryl chloride.To go up 5 ' of nucleosides and import phosphate, need before phosphorylation reaction, 2 ' and 3 ' hydroxyl of ribose on the protection nucleosides.After phosphorylation is finished, slough protective material again, obtain 5 '-Nucleotide.
Because the chemical synthesis agents useful for same is expensive, causes production cost higher, and has certain toxicity, generally is used to produce some Nucleotide that special purpose is arranged and derivatives thereof.
The history that enzymolysis process is produced 5 '-Nucleotide is the longest, technology is also the most ripe.Utilize the Penicillium citrinum fermentation, obtain 5 '-phosphodiesterase and react, can generate the mixture of four kinds of 5 '-Nucleotide, mixture through the ion exchange resin separation and purification, can be obtained the pure product of four kinds of Nucleotide with the RNA that from yeast, extracts.
Usually adopt solid fermentation or tank fermentation method preparation 5 '-phosphodiesterase.The former is easy, cost is low, but floor space is big, and production efficiency is low, and the easy contaminate environment of conidium that produces, and latter's cost is higher.After utilizing 5 '-phosphodiesterase enzymolysis RNA, four kinds of mixture of ribonucleotides need be carried out separation and purification, just can obtain the pure product of 5 '-t-inosinic acid.But obtaining 5 '-IMP, then is that 5 '-AMP is carried out chemistry or enzymatic conversion, complex manufacturing, and cost is higher.
Early stage 5 '-CMP (5 '-cytidylic acid), the 5 '-UMP (5 '-uridylic acid) irrelevant that obtain with enzyme process with being flavor, can't utilize at that time, and make cost higher, therefore, study on the successful basis at glutamic acid fermentation, Japan has begun the fermentation research of nucleic acid material.
Microbe fermentation method divides single stage method and two step method again.Single stage method is mainly used in the production of 5 '-t-inosinic acid and 5 '-xanthylic acid(XMP).Two step method promptly directly generates nucleosides by carbon source through fermentation production or fermentation earlier, again through chemical phosphorylation, has obtained corresponding Nucleotide.
1961, Japanese scientist utilized the VITAMIN B4 defective type of subtilis (Bacillus subitilis) to generate 5 '-t-inosinic acid, inosine.Reported the subtilis VITAMIN B4 deficient strain of high yield inosine subsequently again, and set up inosine suitability for industrialized production (Konjo K, Imai K, et al.Annual Congress Agr.Chem.Soc.1963 Abstracts, P.40).Nineteen sixty-eight when the Brevibacterium ammoniagenes (B.Ammoniagenes) of further investigation VITAMIN B4 defective type, adds mn ion in substratum, to observe the influence to its growth, the result has screened the insensitive bacterial strain of mn ion, can the high yield t-inosinic acid.Above-mentioned result of study shows that single stage method is produced the seed selection more complicated of bacterial strain, and the t-inosinic acid of generation is easy to decompose, and solves or remove also more complicated of end product feedback regulation problem, and this has all greatly limited the industrial application that single stage method is produced t-inosinic acid; And the intermediate product that two step method obtains itself also is very important leavened prod as inosine, guanosine etc., and its regulation and control are relatively easy, so after the key issue of two step method production technique is the intermediate product fermentation, how to change into target Nucleotide efficiently.
Discoveries such as Japanese Asano in 1999, the acid phosphatase of many enterobacterias such as Morganella morganii has selectivity Phosphoric acid esterase transferase active.This Phosphoric acid esterase can utilize tetra-sodium and Nucleotide to be substrate, the generation of specificity catalysis 5 ' t-inosinic acid, thus the 5 ' phosphorylation of carrying out nucleosides for enzyme process provides novel method.And, aspect nucleoside phosphorylaseization,, represented bigger development potentiality because enzyme catalysis has reaction conditions gentleness, low cost, hypotoxicity and specificity advantages of higher.Yet up to now, the parameter of the relevant catalytic process of zymetology kinetics, cell catalysis, fusion rotein catalysis and the enzyme of relevant acid phosphatase transferase active was not also reported by system.
By above analysis as seen, the method for above-mentioned synthetic 5 ' flavour nucleotide respectively has deficiency, and the novel method that Development and Production is the agent of distinguishing the flavor of is one of important directions of Nucleotide fermentation industry development.At present, China's fermentative Production inosine and guanosine have been realized industrialization, and further Development and Production 5 '-key of t-inosinic acid technology is how to realize the specificity phosphorylation of inosine and guanosine based on this.Enzymatic conversion method has very big development potentiality because of it has advantages such as simple and effective and selectivity are strong, and exploitation has the bacterial strain and the technology of synthetic 5 ' the flavour nucleotide potentiality of specificity catalysis, is the key of dealing with problems.
Summary of the invention
The method that the purpose of this invention is to provide a kind of output biosynthesizing 5 ' flavour nucleotide higher, with low cost.
For achieving the above object, the present invention takes following technical scheme: a kind of method of biosynthesizing 5 ' flavour nucleotide, be that enteroaerogen (Enterobacter aerogenes) the IAM1183 thalline that will contain acid phosphatase gene adds in the reaction soln that contains substrate tetra-sodium and inosine (Inosine) or guanosine (Guanosine) as catalyzer, at 25-35 ℃, pH 4.0-8.4 is reaction down, obtains 5 '-t-inosinic acid (5 '-IMP) or 5 '-guanylic acid (5 '-GMP).
In the synthetic method of above-mentioned 5 ' flavour nucleotide, reaction conditions is preferably 30 ℃, and pH 4.6.
For obtaining higher combined coefficient, can carry out above-mentioned catalyzed reaction with smudge cells liquid with after containing the enteroaerogen somatic cells fragmentation of acid phosphatase gene, the reaction conditions of this moment is preferably 30 ℃, and pH 7.6.
Available ordinary method is carried out break process to the enteroaerogen somatic cells, as ultrasonic, chemically fragmenting method or zymetology crush method etc.The method of described broken thalline can be: thalline is placed pH 7.2-8.4, and in the TrisHCl damping fluid of 0.1mM, in 0 ℃ of ice-water bath ultrasonic 50-150 time, each ultrasonic time is 2-4sec, and be 2-4sec pitch time.
The concentration of inosine is 5-40mg/mL in the above-mentioned reaction solution, is preferably 10mg/mL; The concentration of guanosine is 5-40mg/mL, is preferably 10mg/mL; The concentration of tetra-sodium is 200-300mg/mL, is preferably 250mg/mL; The concentration of enteroaerogen somatic cells is 10-80mg/mL, is preferably 20mg/mL.
In addition, for improving enzymatic reaction efficient, in above-mentioned reaction solution, also can add the sal epsom that concentration is 0.1-0.4mg/mL, so that Mg to be provided
2+
The described enteroaerogen that contains acid phosphatase gene can obtain according to conventional cultural method, and as enteroaerogen being inoculated in the LB liquid nutrient medium, at 30-37 ℃, 100-250rpm cultivates down.
The invention provides a kind of biosynthesizing flavour nucleotide (5 '-method of IMP and 5 '-GMP).This method is to be substrate with tetra-sodium and inosine (or guanosine), utilize activity of acid phosphatase that enteroaerogen has the catalysis of inosine (or guanosine) specificity is generated 5 '-IMP (or 5 '-GMP), reaction principle is seen Fig. 1.The present invention has the following advantages:
1) bacterial strain of selecting for use has the activity of acid phosphatase height, and the advantage that fast growth and environmental compatibility are strong all can be grown under aerobic and anaerobic condition, is suitable for suitability for industrialized production;
2) synthetic method is simple, the output height of purpose product, 5 '-concentration that the concentration of t-inosinic acid can reach 6.21mg/mL, 5 '-guanylic acid can reach 6.0mg/mL;
3) raw material sources are extensive, and are low for equipment requirements, low production cost.
The present invention will play a significant role in the suitability for industrialized production of 5 ' flavour nucleotide, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is the schematic diagram of the present invention 5 '-t-inosinic acid synthetic method.
Fig. 2 for the pH value to the enteroaerogen intact cell catalysis synthetic 5 '-detected result of t-inosinic acid influence.
Fig. 3 be optimum pH enteroaerogen intact cell catalysis synthetic 5 '-the t-inosinic acid detected result of variation relation in time.
Fig. 4 for the enteroaerogen intact cell catalysis synthetic 5 '-transformation efficiency of substrate inosine and target product 5 during t-inosinic acid '-transformation efficiency of the t-inosinic acid detected result of variation relation in time.
Fig. 5 for carry out with enteroaerogen cytoclasis liquid catalysis 5 '-different pH enzyme activities influence during the biosynthesizing of t-inosinic acid detected result.
When Fig. 6 is optimum pH the catalysis of enteroaerogen cytoclasis liquid synthetic 5 '-the t-inosinic acid detected result of variation relation in time.
Fig. 7 be the catalysis of enteroaerogen cytoclasis liquid synthetic 5 '-transformation efficiency of substrate inosine and target product 5 during t-inosinic acid '-transformation efficiency of the t-inosinic acid detected result of variation relation in time.
Fig. 8 is the existing way detected result of enteroaerogen inner acidic phosphotransferase.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
1, the acquisition of enteroaerogen (Enterobacter aerogenes) IAM1183 thalline
Single bacterium colony of picking enteroaerogen (Enterobacter aerogenes) IAM1183 (available from using microbe institute of Tokyo Univ Japan (IAM) strain library), it is inoculated in 50mL fills in the little triangular flask of 20mL LB liquid nutrient medium, cultivate 12 hours (spending the night) as seed liquor at 37 ℃, 100rpm shaking table; Get the 1.5mL seed liquor again, it is inoculated in the 250mL triangular flask that fills 150mL LB liquid nutrient medium, under 37 ℃, 180rpm, be cultured to OD
600Value is 1.8 o'clock, and the centrifugal 8min of 12000rpm abandons supernatant, obtains enteroaerogen (Enterobacter aerogenes) IAM1183 somatic cells.
2, enteroaerogen (Enterobacter aerogenes) IAM1183 intact cell catalysis 5 '-during the biosynthesizing of t-inosinic acid different pH to intact cell catalysis synthetic 5 '-influence of t-inosinic acid
To containing the 10mg/mL inosine, 250mg/mL Na
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 0.4g enteroaerogen (Enterobacter aerogenes) the IAM1183 somatic cells (quite 20mg/mL) that step 1 obtains in the solution reaction system of O, then at 30 ℃ and different pH (4.0-5.6) down behind the reaction 16h, the centrifuging and taking supernatant, dilute one times, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid carries out qualitative, quantitative assay, with detect different pH to enteroaerogen (Enterobacter aerogenes) IAM1183 intact cell catalysis synthetic 5 '-influence of t-inosinic acid.Detected result as shown in Figure 2 (ordinate zou is 5 '-concentration (mg/mL) of t-inosinic acid, X-coordinate is the pH value), the pH value all can generate 5 during for 4.0-5.6 '-t-inosinic acid, and be about at 4.8 o'clock in the pH value, 5 '-synthesis rate of t-inosinic acid is the fastest.
Obtain the enteroaerogen somatic cells with the method identical, then to containing the 10mg/mL inosine, 250mg/mL Na with embodiment 1
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 0.4g enteroaerogen (Enterobacter aerogenes) the IAM1183 somatic cells (quite 20mg/mL) that embodiment 1 step 1 obtains in the solution of O, under 28 ℃, pH4.8, carry out catalyzed reaction then, every sampling in 2 hours, the centrifuging and taking supernatant, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid carries out qualitative, quantitative assay, with detect enteroaerogen (Enterobacteraerogenes) IAM1183 intact cell catalysis synthetic 5 '-t-inosinic acid concerns over time.Detected result as shown in Figure 3 (ordinate zou is 5 '-concentration (mg/mL) of t-inosinic acid, X-coordinate is the reaction times), prolongation along with the reaction times, 5 '-concentration of t-inosinic acid is approximate to be linear and to increase, show that speed of response there is no obvious decline, promptly concentration of substrate is excessive relatively, and speed of response still keeps top speed, the phosphotransferase of enteroaerogen (Enterobacter aerogenes) IAM1183 still has higher vigor, and enzyme is lived stable.
Embodiment 3, with the whole cell of enteroaerogen (Enterobacter aerogenes) IAM1183 carry out 5 '-biosynthesizing of t-inosinic acid and substrate conversion efficiency and target product transformation efficiency concern over time
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 somatic cells with the method identical, then to containing the 8mg/mL inosine, 300mg/mL Na with embodiment 1
4P
2O
710H
2O and 0.4mg/mL MgSO
47H
2Add 0.4g enteroaerogen (Enterobacter aerogenes) the IAM1183 cell (quite 20mg/mL) that embodiment 1 step 1 obtains in the solution of O, under 30 ℃, pH4.8, carry out catalyzed reaction then, every sampling in 2 hours, the centrifuging and taking supernatant, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid and concentration of substrate carry out qualitative, quantitative assay.With the transformation efficiency that detects the substrate inosine and target product 5 '-transformation efficiency of t-inosinic acid concerns over time.(ordinate zou is transformation efficiency (%) to detected result as shown in Figure 4, X-coordinate is the reaction times), the inosine transformation efficiency concerns comparatively mild over time, and is little with the variation in reaction times, show that inosine is stronger for the cell membrane permeability, can more easily enter cell; And 5 '-generation of t-inosinic acid change in time comparatively obvious, and approximately linear, this with 5 '-perviousness of t-inosinic acid is relatively poor relevant, synthetic 5 '-t-inosinic acid need progressively be discharged into outside the born of the same parents.
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical with embodiment 1, use the broken somatic cells of ultrasonic method then, method is: thalline is placed pH 7.6,0.1mM the TrisHCl damping fluid in, in 0 ℃ of ice-water bath ultrasonic 99 times, each ultrasonic time is 3sec, and be 3sec pitch time.Again to containing the 10mg/mL inosine, 250mg/mL Na
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 20mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 bacterial cell disruption liquid in the solution of O, then at 30 ℃ and different pH (5.2-8.4) down behind the reaction 10min, with 0.15mL concentrated hydrochloric acid termination reaction.With high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid carries out qualitative, quantitative assay.(ordinate zou is a free phosphoric acid transferring enzyme vigor (U) to the enzymic activity detected result as shown in Figure 5, MES represents sodium acetate buffer pH value of solution 5.2-6.8, TrisHCl represents: the pH7.2-8.4 X-coordinate is the pH value), at pH is 7.6 o'clock, free phosphoric acid transferring enzyme vigor is the highest, and have maximum catalyzed reaction speed, be the pH value of optimum response, also illustrate that pH value is more obvious when putting in order cell with enteroaerogen and carry out catalysis and synthesize to the effect of vigor of free phosphoric acid transferring enzyme simultaneously.
The method identical with embodiment 1 obtains enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell, uses the method smudge cells identical with embodiment 4 then, again to containing 15mg/mL inosine, 250mg/mLNa
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 40mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 bacterial cell disruption liquid in the solution of O, under 30 ℃, pH 7.6 conditions, carry out catalyzed reaction then,, add 0.15mL concentrated hydrochloric acid termination reaction every sampling in 2 hours.Centrifugal 20min under 20 ℃, 8000rmp, with 1 times of supernatant liquor dilution, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid carries out qualitative, quantitative assay; With detect the catalysis of enteroaerogen (Enterobacter aerogenes) IAM1183 smudge cells synthetic 5 '-t-inosinic acid concerns over time.Detected result as shown in Figure 6 (ordinate zou is 5 '-concentration (mg/mL) of t-inosinic acid, X-coordinate is the reaction times), the smudge cells catalytic capability is stronger, react 10 minutes can with add the inosine total amount 38.4% be converted into 5 '-t-inosinic acid, when 5 '-when t-inosinic acid concentration reaches 5.8mg/mL, speed of response obviously descends, and illustrates that enzyme work is subjected to the inhibition of product.
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical, use the method broken somatic cells identical then, again to containing 10mg/mL inosine, 250mg/mLNa with embodiment 4 with embodiment 1
4P
2O
710H
2O and 0.1mg/mL MgSO
47H
2Add 15mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 bacterial cell disruption liquid in the solution of O, under 30 ℃, pH7.6, carry out catalyzed reaction then, every 2 hours, with 0.15mL concentrated hydrochloric acid termination reaction.With high pressure lipuid chromatography (HPLC) to synthetic 5 '-t-inosinic acid and concentration of substrate carry out qualitative, quantitative assay, with the transformation efficiency that detects the substrate inosine and target product 5 '-transformation efficiency of t-inosinic acid concerns over time.(ordinate zou is transformation efficiency (%) to detected result as shown in Figure 7, X-coordinate is the reaction times), in the initial short time, reaction can be carried out soon, in 10min, 5 '-concentration of t-inosinic acid can reach 5.85mg/mL, after this 5 and '-concentration of t-inosinic acid is with the variation in reaction times and not obvious, after arriving 16h, 5 '-concentration of t-inosinic acid can reach 6.21mg/mL.
The existing way of embodiment 7, detection enteroaerogen (Enterobacter aerogenes) IAM1183 inner acidic phosphotransferase
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical with embodiment 1, use the method smudge cells identical then with embodiment 4, centrifugal 20min under 20 ℃, 8000rmp then, separation of supernatant and precipitation, again with the supernatant liquor and the precipitation of broken thalline, and the cultivation bacterium liquid that does not carry out the broken thalline of centrifugation and contain whole cell is added to as catalytic materials respectively and contains the 10mg/mL inosine, 250mg/mL Na
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Under 30 ℃, pH 7.6 conditions, carry out catalyzed reaction in the solution of O, with the negative contrast of the reaction system of no catalytic materials, with 5 of high-pressure liquid phase chromatogram therapy determining product '-concentration of t-inosinic acid.Detected result as shown in Figure 8 (ordinate zou is 5 '-concentration (mg/mL) of t-inosinic acid, X-coordinate is the reaction times), the catalytic capability of enteroaerogen cytoclasis liquid supernatant is much larger than the sedimentary catalytic capability of fragmentation, the acid phosphatase transferring enzyme that enteroaerogen (Enterobacter aerogenes) IAM1183 is described is in the cell, and has the strong characteristics of solubility.
Obtain enteroaerogen (Enterbacter aerogenes IAM1183) whole cell with the method identical, then to containing the 10mg/mL guanosine, 250mg/mL Na with embodiment 1
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 20mg/mL enteroaerogen (Enterbacter aerogenes IAM1183) whole cell in the solution of O, under 30 ℃, pH4.8, carry out catalyzed reaction then, and every sampling in 2 hours, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid carries out qualitative, quantitative assay.The result is along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, and shows that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Embodiment 9, utilize the enteroaerogen smudge cells synthetic 5 '-guanylic acid
Obtain enteroaerogen (Enterbacter aerogenes IAM1183) whole cell with the method identical, use the method smudge cells identical then, again to containing 12mg/mL guanosine, 280mg/mLNa with embodiment 4 with embodiment 1
4P
2O
710H
2O and 0.3mg/mL MgSO
47H
2Add 50mg/mL enteroaerogen (Enterbacter aerogenes IAM1183) cytoclasis liquid in the solution of O, carry out catalyzed reaction 8.0 times at 32 ℃, pH then, every 2 hours with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid concentration carries out qualitative, quantitative assay.Result and along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, and shows that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical, then to containing the 10mg/mL guanosine, 250mg/mL Na with embodiment 1
4P
2O
710H
2O and 0.2mg/mL MgSO
47H
2Add 20mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell in the solution of O, under 30 ℃, pH4.8, carry out catalyzed reaction then, and every sampling in 2 hours, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid carries out qualitative, quantitative assay.The result is along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, arrive 16h after, 5 '-concentration of guanylic acid can reach 5.9mg/mL, show that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Embodiment 11, utilize the whole cell of enteroaerogen (Enterobacter aerogenes) IAM1183 synthetic 5 '-guanylic acid
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical, then to containing 5mg/mL guanosine and 200mg/mL Na with embodiment 1
4P
2O
710H
2Add 10mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell in the solution of O, under 25 ℃, pH7.6, carry out catalyzed reaction then, and every sampling in 2 hours, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid carries out qualitative, quantitative assay.The result is along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, 5 '-concentration of guanylic acid can reach 5.5mg/mL, shows that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical, then to containing the 40mg/mL guanosine, 300mg/mL Na with embodiment 1
4P
2O
710H
2O and 0.4mg/mL MgSO
47H
2Add 80mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell in the solution of O, under 35 ℃, pH8.4, carry out catalyzed reaction then, and every sampling in 2 hours, with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid carries out qualitative, quantitative assay.The result is along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, 5 '-concentration of guanylic acid can reach 5.9mg/mL, shows that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Embodiment 13, utilize the enteroaerogen smudge cells synthetic 5 '-guanylic acid
Obtain enteroaerogen (Enterobacter aerogenes) IAM1183 whole cell with the method identical with embodiment 1, use the broken somatic cells of ultrasonic method then, method is: thalline is placed pH 8.4,0.1mM the TrisHCl damping fluid in, in 0 ℃ of ice-water bath ultrasonic 150 times, each ultrasonic time is 5sec, and be 2sec pitch time.Again to containing the 12mg/mL guanosine, 280mg/mL Na
4P
2O
710H
2O and 0.1mg/mL MgSO
47H
2Add 50mg/mL enteroaerogen (Enterobacter aerogenes) IAM1183 cytoclasis liquid in the solution of O, carry out catalyzed reaction 7.6 times at 30 ℃, pH then, every 2 hours with high pressure lipuid chromatography (HPLC) to synthetic 5 '-guanylic acid concentration carries out qualitative, quantitative assay.Result and along with the prolongation in reaction times, 5 '-concentration of guanylic acid is approximate to be linear and to increase, 5 '-concentration of guanylic acid can reach 6.0mg/mL, shows that the selected bacterial strain of the present invention has higher activity of acid phosphatase, can be used for 5 '-biosynthesizing of guanylic acid in.
Claims (7)
1, the method for a kind of biosynthesizing 5 '-t-inosinic acid or 5 '-guanylic acid, be that enteroaerogen (Enterobacter aerogenes) the IAM1183 thalline that will contain acid phosphatase gene adds in the reaction soln that contains substrate tetra-sodium and inosine or guanosine, at 25-35 ℃, pH 4.0-8.4 is reaction down, obtains 5 '-t-inosinic acid or 5 '-guanylic acid; Be added with the sal epsom that concentration is 0.1-0.4mg/mL in the described reaction soln.
2, method according to claim 1 is characterized in that: described reaction conditions is 30 ℃, pH4.6.
3, method according to claim 1 is characterized in that: the enteroaerogen somatic cells that fragmentation contains acid phosphatase gene, react with cytoclasis liquid again.
4, method according to claim 3 is characterized in that: the method for described broken thalline is: thalline is placed pH7.2-8.4, and in the TrisHCl damping fluid of 0.1mM, in 0 ℃ of ice-water bath ultrasonic 50-150 time, each ultrasonic time is 2-4sec.Be 2-4sec pitch time.
5, method according to claim 3 is characterized in that: described reaction conditions is 30 ℃, pH7.6.
6, according to each described method of claim 1-5, it is characterized in that: the concentration of described inosine is 5-40mg/mL, and the concentration of guanosine is 5-40mg/mL, and the concentration of tetra-sodium is 200-300mg/mL, and the addition of enteroaerogen thalline is 10-80mg/mL.
7, method according to claim 6 is characterized in that: the concentration of described inosine is 10mg/mL, and the concentration of guanosine is 10mg/mL, and the concentration of tetra-sodium is 250mg/mL, and the addition of enteroaerogen thalline is 20mg/mL.
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