CN1670207A - Immobilized nucleoside phosphorylase microbe and its application in synthesizing purine nucleosidase - Google Patents
Immobilized nucleoside phosphorylase microbe and its application in synthesizing purine nucleosidase Download PDFInfo
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Abstract
This invention discloses a method for immobilizing riboside phosphatase and its application in the course of synthesizing purine nucleosides. The method has the advantages of operation simplicity, good reutilization character, non-destroyed carrier, short reaction time, high percent conversion of enzyme. The invention is characterized in adopting 40mmol/L Ade or 2, 6-diaminopurine as acceptor of ribose or deoxyribose, 40mmol/L thymidine or inosinic as donator of ribose or deoxyribose to fix acetyl brevibacterium as enzyme accelerant; acquiring rate conversion high than 60%(mol) through two-hour reaction in condition of phosphate; immobilization microbios being repeatedly-usable for 15-20 times, and well applied in industrial manufacture.
Description
Technical field
The present invention relates to immobilization and contain nucleoside phosphorylase microbe and the application in the purine biosynthesis nucleosides thereof.
Background technology
Nucleoside compound is the many important antiviral antitumor nucleotide medicines and the intermediate of other biochemical reagents.Wherein adenosine can be used for closing vidarabine and adenosine triphosphate; 2,6-diaminopurine nucleosides DAPR can be used as biochemical reagents and is used for molecular biology research; 2 '-Desoxyadenosine dAR is the important intermediate of Synthetic 2-chlorine Desoxyadenosine; 2, the fast deoxynucleoside DAPdR of 6-diamino is a Synthetic 2 '-important intermediate of pancreatic desoxyribonuclease.
The preparation of nucleosides generally can be obtained by following method:
A.RNA or DNA hydrolysis method
The initial method that obtains deoxidation or non-deoxynucleoside is by crude substance such as hydrolysis RNA or DNA, obtains the mixture of multiple nucleosides, therefrom isolates required nucleosides then.For example, be raw material with the calf thymus thymus nucleic acid, after deoxyribonuclease (or phosphodiesterase) and phosphoesterase hydrolysis, column chromatography for separation is refining can to get 2 '-Desoxyadenosine, 2 '-pancreatic desoxyribonuclease, 2 '-Deoxyribose cytidine and thymidine; With yeast rna similarly method obtain four kinds of natural not deoxynucleosides.But this method has many shortcomings, because the amount that contains DNA or RNA in the various natural biologicals is very limited, and the degraded back is the mixture of multiple nucleosides, separation and Extraction is very complicated, therefore limited the large-scale industrial production nucleosides, and the nucleosides of gained only is a natural nucleus glycoside.
B. fermentation method
The five kinds of natural nucleus glycosides (adenosine, inosine, uridine, cytidine, guanosine) of not deoxidation of fermentative Production that in recent years a lot of bibliographical informations arranged, and obtained very big progress.Wherein inosine and guanosine can be realized suitability for industrialized production, but for adenosine, have serious instability owing to produce bacterial classification, have limited the application in the industry greatly.
C. chemical synthesis
Most nucleoside all can obtain by full chemosynthesis, as some important anti-AIDS drug zidovudines, stavudine and some antitumor drugs such as Furtulon etc.Protection be protected and be gone to chemical synthesis need to the active group of base and ribosyl; And there is the extremely difficult removal fully from the finished product of optical isomer, influences ultimate yield; Also need in the technological process to use a large amount of poisonous and hazardous organic solvents, environmental pollution is also dangerous; Some purine class deoxynucleoside can't synthesize with existing method as 2 '-Desoxyadenosine and 2 '-pancreatic desoxyribonuclease etc.
D. enzyme transforming process
A lot of bibliographical informations utilize nucleoside phosphorylase or N-ribodesose transferring enzyme enzyme process synthetic nucleosides compounds, detail as per document 1: " Boorganic Chemistry; 1999; 27:135-154 ", document 2: " Journal of Molecular Catalysis B:Enzymatic.1999; 6 (3): 215-222 ", document 3: " Chinese Journal of Pharmaceuticals, 1999; 30 (10): 474-478 " etc.
Document wherein: " Biocatalysis and Biotransformation; 2002; 20 (5): 347-351 " reported with Escherichia coli BL21 and done the catalyst Synthetic 2 '-deoxynucleoside, the transformation efficiency of 2 '-Desoxyadenosine has been issued to 94% at 45 ℃, but the concentration of used uridine and VITAMIN B4 is respectively 30mM and 10mM, and temperature of reaction neither be very high, be unfavorable for the dissolving of substrate, therefore the final concentration of the target product that obtains is lower, that is industrial throughput is lower, what it adopted in addition is free thalline, is not suitable for repeatedly reusing.In another piece document " Journal of MolecularCatalysis B:Enzymatic 10 (2000) 207-213 ", also reported the synthetic of 2 '-deoxynucleoside, do the enzyme source with Enterobacter aerogenes, wherein 2 '-Desoxyadenosine can obtain higher transformation efficiency under higher concentration, but, there are unfavorable factors such as the easy inactivation of enzyme owing to use free thalline.Document " Journal of Molecular Catalysis B:Enzymatic 30 (2004) 219-227 " has been reported with polyacrylamide, alginate calcium, agar and agarose as Immohilized microorganism carrier, can the enzyme process high conversion syntheticly obtain various purine class nucleosides, wherein polyacrylamide immobilization Citrobacteramalonaticus CECT863 can reuse more than 50 times, but because of used concentration of substrate too low, the VITAMIN B4 of having only uridine and the 0.05mmol/L of 0.15mmol/L is almost without any practical value.
The technical issues that need to address of the present invention are to disclose a kind of immobilization nucleoside phosphatase animalculum and the application in the purine biosynthesis nucleosides thereof, to overcome the shortcoming that above technology exists.
Design of the present invention is such:
Employing has the wet thallus of high vigor Phosphatase, nucleotide microorganism, then with kappa-carrageenan etc. as fixation support, being fixed thalline directly carries out enzymic catalytic reaction again, obtains target product.Its principle is to utilize the interior purine nucleoside phosphorylase of born of the same parents and the pyrimidine-nucleoside phosphorylase of microorganisms to carry out enzymic catalytic reaction.The nucleosides that is easy to get is changed into the target nucleosides.
Immobilization nucleoside phosphatase animalculum of the present invention is made of nucleoside phosphorylase microbe and fixation support, and wherein, the content of nucleoside phosphorylase microbe wet thallus is with respect to 10～30% of the weight of the immobilization particle for preparing;
Said wet thallus is meant being in the microorganism in logarithmic growth later stage, 10, carries out centrifugal 20min under the rotating speed of 000r/min, the part of anhydrating of inclining, and the bacterium mud of lower floor can be standby.
Said nucleoside phosphorylase microbe is selected from intestinal bacteria, enteroaerogen, Hu Luobu soft rot Erwinia, acetyl-tyrothricin or mycobacterium lacticolum etc., these microorganisms can be available from each culture presevation mechanism, or obtain by certain procedure from occurring in nature, preferred microorganism is that acetyl-tyrothricin (Brevibacterium acetylium) QD96 comes from Chinese microorganism strain preservation administrative center, and preserving number is: CGMCC No.0472;
Used fixation support is selected from one or more in alginate calcium, carrageenin, agarose, polyacrylamide, polyvinyl alcohol or the collagen protein etc., and preferred carrageenin or polyacrylamide are wherein with the carrageenin best results;
The preparation method of immobilized nucleoside phosphorylase microbe of the present invention is very simple, as adopting carrageenin, can adopt entrapping method, comprises the steps:
Carrageenin is added entry, stir evenly swelling in the boiling water bath after, standby in 40 ℃ of-45 ℃ of insulations;
Centrifugal collection is in the nucleoside phosphorylase microbe wet thallus in logarithmic phase later stage, behind the physiological saline mixing of adding equivalent, is warming up to 40 ℃-45 ℃, and the reserve liquid with above-mentioned carrageenin carrier mixes then; After stirring, pour in the dish, be paved into the film of 2-4mm, place 4 ℃ of refrigerator cooling 2h again, be cut into the small-particle of 2-4mm afterwards, be soaked in 3% KCl solution 2 hours, wash twice with 3% KCl again, be soaked in 1% glutaraldehyde solution again 1 hour, with the phosphate buffered saline buffer washing of pH7.0,50mmol/L three times, drop anhydrates part (above operation is all being carried out below 10 ℃), and sealed storage is standby in 4 ℃ of refrigerators then;
As adopt polyacrylamide, comprise following step:
In 29ml physiological saline, dissolve in the two third rare acid amides of 4.5g third rare acid amides and 0.5g methene, add the 10g wet thallus then, stir evenly, add 5% β-dimethylamino propionitrile and 2.5% K again
2S
2O
8Each 3ml, thus the polyacrylamide gel formed.With cutter gel is cut into the particle of 3mm, soaks 30min, wash twice with the potassium phosphate buffer of pH7.0,50mmol/L again with 1% glutaraldehyde, the drop part (above operation is all being carried out below 10 ℃) of anhydrating, sealed storage is standby in 4 ℃ of refrigerators then.
Adopt the immobilization nucleoside phosphatase animalculum of method for preparing, can be used for preparing nucleoside compound, as adenosine, 2,6-diaminopurine nucleosides, 2 '-Desoxyadenosine, 2, the fast deoxynucleoside of 6-diamino.
The method that adopts the immobilized nucleoside phosphorylase microbe of being addressed to prepare nucleoside compound comprises the steps:
Said fixing nucleoside phosphorylase microbe and ribose donor and ribose acceptor are carried out enzymic catalytic reaction in potassium phosphate buffer, pH is 6.5～7.5, temperature of reaction is 45～55 ℃, reaction times is 1～3h, the ribose donor that contains 30～50mmol/L in the reaction solution of 10mL, the ribose acceptor of 30～50mmol/L, in the above-mentioned reaction system, the consumption of immobilization nucleoside phosphatase animalculum is equivalent to the 0.5g wet thallus, the consumption of potassium phosphate buffer is 40～60mmol/L, collects nucleoside compound then from reaction product.
Said ribose acceptor is selected from VITAMIN B4,2,6-diaminopurine or xanthoglobulin;
Said ribose donor is selected from and is inosine, uridine, cytidine, 5-methyluridine, thymidine, deoxyuridine or Deoxyribose cytidine etc.
Immobilization nucleoside phosphatase animalculum of the present invention can obtain higher transformation efficiency and enzyme stability preferably.Use above process for fixation fixed acetyl-tyrothricin QD96, being more or less the same of the transformation efficiency of its enzyme reaction and free acetyl-tyrothricin.At concentration of substrate is 40mmol/L, and temperature is 50 ℃, and during the potassium phosphate buffer 50mmol/L of pH7, its transformation efficiency can both reach more than 60%.
The immobilized bacterium precursor reactant reaches the required time needs of balance two hours (free thalline is 1h), and this may exist due to the mass transfer obstacle, but this does not influence its application.Immobilized acetyl-tyrothricin recycling number of times also is that its enzyme stability obviously improves apparently higher than free thalline.The free acetyl-tyrothricin is after recycling 3 times, nucleoside phosphorylase in its born of the same parents is inactivation substantially, even in above-mentioned 3 recycling processes, all reduce each time than the transformation efficiency of last time, so free acetyl-tyrothricin and be not suitable for repeatedly reusing.Immobilized acetyl-tyrothricin can reuse about 20 times basically, and the enzyme reaction transformation efficiency reduces less between per twice.Therefore, this helps saving the required cost of cultivation acetyl-tyrothricin.
Said transformation efficiency is to measure by the HPLC method.Method is as follows:
Island body fluid chromatography; Post YWG-C18 (200mm * 4.6mm); Moving phase: the phosphate buffered saline buffer of 5% acetonitrile+50mmol/L pH7.0; Flow velocity: 1.0ml/min; Wavelength: 254nm.
Description of drawings
Fig. 1 is carrageeenen immobilized thalline Synthetic 2 '-the reaction repeated curve of Desoxyadenosine.
Fig. 2 is a polyacrylamide immobilization thalline Synthetic 2 '-the reaction repeated curve of Desoxyadenosine.
Fig. 3 is the synthetic DAPdR response curve of applying immobilized thalline.
Fig. 4 is the synthetic DAPR response curve of immobilized cell.
Fig. 5 is an immobilized cell synthesizing adenosine response curve.
Embodiment
Embodiment 1
The 1.2g carrageenin is added in the 28.8mL deionized water, and swelling in boiling water bath places 40 ℃ of water-baths to be incubated then again.Get 10g acetyl-tyrothricin wet thallus, add 10mL physiological saline and stir evenly, heat temperature raising in 40 ℃ of water-baths then, treat that temperature reaches 40 ℃ after, mix with above-mentioned carrageenin insulation liquid immediately, stir evenly.Pour in the dish, be paved into the film of 2-4mm, be positioned in 4 ℃ of refrigerators and solidify 2h, be cut into the particle of 3mm again, these particles are soaked 2h in 3% KCl solution after, wash twice with 3% KCl again, be soaked in 1% glutaraldehyde solution again 1 hour, phosphate buffered saline buffer with pH7.0,50mM washs three times, and drop anhydrates part (above operation is all being carried out below 10 ℃), and the thalline that fixes can be preserved standby in 4 ℃ of refrigerators.
Get the immobilized thalline of 2.5g (being equivalent to the 0.5g wet thallus) and carry out enzymic catalytic reaction, reaction is carried out under the following conditions: contain the thymidine (Thymidine) of 40mmol/L, the VITAMIN B4 of 40mmol/L and the potassium phosphate buffer 50mmol/L of pH7.0 in the reaction solution of 10mL, temperature of reaction is 50 ℃, and the time is 2h.Reacted the back and collected immobilized thallus, washed twice with above-mentioned potassium phosphate buffer again, repeated above-mentioned reaction then with filtered through gauze.Reaction finishes the back and goes out thalline in 10000r/min is centrifugal, the results are shown in Figure 1.Among the figure, curve a is an immobilized cell, and curve b is free thalline.
Embodiment 2
In 29ml physiological saline, dissolve in the two third rare acid amides of 4.5g third rare acid amides and 0.5g methene, add the 10g wet thallus then, stir evenly, add 5% β-dimethylamino propionitrile and 2.5% K again
2S
2O
8Each 3ml, thus the polyacrylamide gel formed.With cutter gel is cut into the particle of 3mm, soaks 30min, wash twice with the potassium phosphate buffer of pH7.0 again with 1% glutaraldehyde, the drop part (above operation is all being carried out below 10 ℃) of anhydrating, the immobilized thallus that immobilization is good can be stored in 4 ℃ of refrigerators standby.
Get immobilized thallus 2.5g, react by embodiment 1 described method.The results are shown in Figure 2.
Embodiment 3
Get prepared each 2.5g of immobilized cell of embodiment 1 and embodiment 2, react by following reaction prescription: contain in the reaction solution of 10mL 40mmol/L thymidine (Thymidine), 40mmol/L 2, the potassium phosphate buffer of 6-diaminopurine and pH7.0,50mmol/L, temperature of reaction is 50 ℃, and the time is 2h.Reacted the back and collected immobilized thallus, washed twice with above-mentioned potassium phosphate buffer again, repeated above-mentioned reaction then with filtered through gauze.After each reaction finishes, 2,6-diaminopurine deoxynucleosides (DAPdR) of generating of sampling and measuring all.Other gets wet thallus 0.5g, and by same reaction, but the reaction times is 1h, and reaction finishes the back and goes out thalline, reaction repeated in 10000r/min is centrifugal.The results are shown in Figure 3.
Among the figure: curve 1 is a carrageenin, and curve 2 is a polyacrylamide, and curve 3 is free thalline.
With embodiment 3, but change thymidine into inosine.The same Fig. 4 of result.Among the figure, curve 4 is a carrageenin, and curve 5 is a polyacrylamide, and curve 6 is free thalline.
With embodiment 3, but change thymidine and 2,6-diaminopurine into inosine and VITAMIN B4 respectively.Result such as Fig. 5.Among the figure, curve 7 is a carrageenin, and curve 8 is a polyacrylamide, and curve 9 is free thalline.
Claims (5)
1. an immobilization nucleoside phosphatase animalculum is characterized in that, is made of nucleoside phosphorylase microbe and fixation support, and wherein, the content of nucleoside phosphorylase microbe wet thallus is with respect to 10～30% of the weight of the immobilization particle for preparing;
Said product nucleoside phosphorylase microbe is selected from intestinal bacteria, enteroaerogen, Hu Luobu soft rot Erwinia, acetyl-tyrothricin or mycobacterium lacticolum;
Used fixation support is selected from one or more in alginate calcium, carrageenin, agarose, polyacrylamide, polyvinyl alcohol or the collagen protein.
2. immobilized nucleoside phosphorylase microbe according to claim 1 is characterized in that, nucleoside phosphorylase microbe is selected from acetyl-tyrothricin CGMCC No.0472.
3. immobilized nucleoside phosphorylase microbe according to claim 2 is characterized in that used fixation support is selected from carrageenin or polyacrylamide.
4. according to the application of claim 1,2 or 3 described immobilized nucleoside phosphorylase microbes, it is characterized in that, be used to prepare nucleoside compound, as adenosine, 2,6-diaminopurine nucleosides, 2 '-Desoxyadenosine or 2, the fast deoxynucleoside of 6-diamino.
5. application according to claim 4, it is characterized in that, comprise the steps: immobilized nucleoside phosphorylase microbe and ribose donor and ribose acceptor are carried out enzymic catalytic reaction in potassium phosphate buffer, pH is 6.5～7.5, temperature of reaction is 45～55 ℃, reaction times is 1～3h, the ribose donor that contains 30～50mmol/L in the reaction solution of 10mL, the ribose acceptor of 30～50mmol/L, in the above-mentioned reaction system, the consumption of immobilized nucleoside phosphorylase microbe is equivalent to the 0.5g wet thallus, and the consumption of potassium phosphate buffer is 40～60mM, collects nucleoside compound then from reaction product;
Said ribose acceptor is selected from VITAMIN B4,2,6-diaminopurine or xanthoglobulin;
Said ribose donor is selected from and is inosine, uridine, cytidine, 5-methyluridine, thymidine, deoxyuridine or Deoxyribose cytidine.
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