CN1300301C - 3'-Rhodotorula glutinis fungus of 3'-phosphodiester enzyme and method for preparing 3'-nucleotide - Google Patents

Abstract

The present invention discloses a rhodotorula glutinis strain of 3'-phosphodiesterase, and a method for preparing 3'-nucleotide from the rhodotorula glutinis strain. The strain of the present invention is CGMCC No. 1272. The strain can be conventionally fermented to prepare the QOX3318, and 3'-nucleotide can be prepared from 3'-phosphodiesterase with an enzyme method. The method of the present invention can prepare enzyme liquid with high vitality generally as that of 250 U/mL fermentation liquor, and the vitality is nearly one time than that reported in documents; more than 70% of the vitality of the prepared enzyme liquid can be maintained when the enzyme liquid is made to stand at 70 DEG C for 1.5 hours; the obtained enzyme liquid can be directly used for enzymatic reaction without being purified, and the degradation rate of RNA reaches more than 90%; the reaction liquid is mainly composed of four kinds of 3'-nucleotide according to HPLC analysis. The method of the present invention has the advantages of obviously improved preparation efficiency and greatly simple operation, and thus, the method of the present invention has high economic practicality.

Description

The Rhodotorula glutinis fungus of 3 '-phosphodiesterase and prepare the method for 3 '-Nucleotide

Technical field

The present invention relates to a kind of rhodotorula glutinis (Rhodotorula glutinis) and adopt this strain fermentation to produce the method for 3 '-phosphodiesterase and this enzyme production 3 '-Nucleotide of application.

Background technology

3 '-phosphodiesterase can be with Yeast Nucleic Acid (Ribonulceic acid, RNA) be degraded to specificity 3 '-adenine nucleotide (Adenosine-3 '-monophosphate, 3 '-AMP), 3 '-guanylic acid (Guanosine-3 '-monophosphate, 3 '-GMP), 3 '-cytidylic acid(CMP) (Cytidine-3 '-monophosphate, 3 '-CMP) and 3 '-uridylate (Uridine-3 '-monophosphate, 3 '-UMP), its structure is as follows:

Described 3 '-Nucleotide is important biochemical reagents, can be used for the demand of synthetic RNA in a large number, also is the important as precursors of a lot of nucleic acid drugs simultaneously.

At present, the preparation method of 3 '-Nucleotide can be divided into two classes:

(1) alkaline hydrolysis RNA preparation:

RNA solution is degraded to the mixture of 2 ' and 3 '-Nucleotide under the potassium hydroxide of 0.3mol/L or sodium hydroxide effect.Utilize this eight kinds of Nucleotide dissociation constant differences, under anion-exchange column, separate, and split out the isomers of 2 ' and 3 '-Nucleotide simultaneously.Because the amount of gained 2 '-Nucleotide and 3 '-Nucleotide much at one, this method efficient is very low, and eight kinds of Nucleotide separate simultaneously, operates very difficultly, and yield is also not high, is not suitable for large-scale industrial production.

(2) enzymolysis process preparation:

This is effective, the most most economical method of 3 '-Nucleotide of producing at present.The outer 3 '-phosphodiesterase of the born of the same parents that utilize rhodotorula glutinis to produce can with the RNA specificity be degraded to four kinds of 3 '-Nucleotide, the RNA degradation rate can reach more than 90%, also is mainly 3 '-Nucleotide in the solution.Utilize the dissociation constant difference of these four kinds of Nucleotide again, can be easily with its separation.This method efficient height, cost is low, is suitable for industrialization and gives birth to.

Many bibliographical informations utilize rhodotorula glutinis fermentative production 3 '-phosphodiesterase and use the technology that this enzyme is produced 3 '-Nucleotide.

Nako etc. find first the extracellular enzyme that rhodotorula glutinis produces (document " and Nakao Y.and Ogata K.; Agr.Biol.Chem.; 1963; 27 (7): 499-506); can the RNA enzymolysis be become 3 '-Nucleotide, but 3 '-phosphodiester enzyme activity that bacterial classification produced of report is extremely low single-mindedly, average enzyme work has only the 10U/mL fermented liquid; and the enzyme liquid of gained is extremely unstable, 70 ℃ down several minutes be inactivation.Sun Rongrong also screens a strain rhodotorula Rhodotorula grcilis No.91 (referring to document " Sun Rongrong chemical reagent; 1983; 5 (2): 103-106 "), 28 ℃ of shake-flask culture 96 hours, enzyme liquid vigor can reach 100-130U/mL, and being successfully used to produce in batches 3 '-Nucleotide, the total recovery of 3 '-Nucleotide is about about 30%.

The about 130U/mL fermented liquid of high enzymatic activity of above-mentioned bibliographical information is unfavorable for the production of Nucleotide; Fermentation period is longer, generally needs about 96 hours even 120 hours, and the yield of 3 '-Nucleotide is low.

Summary of the invention

The technical issues that need to address of the present invention provide a kind of rhodotorula glutinis of product heat-resisting high vigor 3 '-phosphodiesterase, and with the outer 3 '-phosphodiesterase degradation of rna preparation of the born of the same parents of this yeast secretary 3 '-Nucleotide, to overcome the defective that above-mentioned technology exists.

Design of the present invention is like this:

Adopt a kind of rhodotorula glutinis, in the fermention medium that is fit to, cultivate 72h, the centrifugal removal thalline of the fermented liquid of gained.To obtain enzyme liquid and RNA 60 ℃ of reactions one hour, RNA can be degraded, obtain 3 '-Nucleotide solution, obtain highly purified four 3 '-Nucleotide through separating.

Said RNA can extract from yeast or geotrichum candidum cell, or directly buys from market.

The microorganism that the present invention is used:

The used microorganism of the present invention is a kind of rhodotorula glutinis (Rhodotorula glutinis) QOX 3318CGMCC No.1272, and (hereinafter to be referred as QOX 3318) has the ability of secretion heat-resisting high vigor 3 '-phosphodiesterase.

Rhodotorula glutinis is distributed widely among air, water, flower, the soil etc., and can therefrom separate and obtain, or buys from each culture presevation mechanism.The used original strain of the present invention is purchased in Chinese industrial microbial strains preservation center, is numbered CICC 1691.

Through CICC1691 is frozen for a long time deeply, uv irradiating, gamma-rays radiation, chemical reagent handle methods such as (as ethyl sulfate, nitrous acid, N-methyl-nitro-N-nitrosoguanidine etc.), ultrasonic wave and carry out mutagenic treatment, can obtain rhodotorula glutinis (Rhodotorula glutinis) QOX 3318.This bacterial classification on December 10th, 2004 in China Committee for Culture Collection of Microorganisms's preservation, the address: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is: CGMCC No.1272.

Said QOX 3318 has following feature:

1, morphological specificity

(1) cell is very little to the greatest extent: 2.3～5.0 * 4.0～10 μ m;

(2) cell shape: avette or spherical;

(3) gemmation, no syngenesis phenomenon does not form ballistospore;

2, growthhabit

Bacterium colony: cultivated four days on 5 ° of wort agar planes, the lawn color is coral red, and smooth surface is glossy, and the quality thickness hardens sometimes, projection, and regular edges is neat, and aseptic silk forms.

3, physiological property

(1) can with nitrate only nitrogen source, hydrolyzable urea;

(2) any carbohydrate of nonfermented;

(3) do not form starch;

(3) do not produce acetate;

(4) can assimilate glucose, maltose, sucrose, melizitose, D-N.F,USP MANNITOL, N.F,USP MANNITOL, D-wood sugar, D-sorbyl alcohol, D-fructose, ethanol and glycerine etc.;

(5) do not assimilate melibiose, sweet and pure (melampyrin), inositol, lactose, tetrahydroxybutane (butantetraol), synanthrin, L-sorbyl alcohol, D-ribose, glucosamine hydrochloride, dextran, rhamnosyl, pyrogallol, salicin, Zulkovsky starch, dextrin, methyl alcohol etc.;

(6) can both grow in the pH2.5-10.0 scope;

(7) growth is optimal temperature: 22-28 ℃;

(8) can secrete the outer heat-resisting high vigor 3 ' of born of the same parents-phosphodiesterase.

The appraisal basis and the above-mentioned test-results that provide according to document " " Yeasts Characteristics and Identification ": 455-456 " show that QOX 3318 belongs to imperfect fungi, gemma guiding principle, Cryptococeales, Rhodotorula, rhodotorula glutinis, but have obviously different with original strain, its main difference part is that growth temperature can not be above 30 ℃, the cycle of producing 3 '-phosphodiesterase obviously shortens, and the enzyme that is produced has good heat endurance.

By the fermentation of routine, can cultivate above-mentioned QOX3318, and utilize 3 ' of its generation-phosphodiesterase Production by Enzymes 3 '-Nucleotide.Be summarized as follows:

Organic nutrients such as said rhodotorula glutinis QOX3318 can carbon source, nitrogenous source, inorganic salt and essential vitamins are cultivated.

Said carbon source be selected from glucose or/and maltose or/and sucrose or/and D-sorbyl alcohol etc.;

Said nitrogenous source be selected from urea or/and ammonium sulfate or/and nitrate or/and yeast extract paste or/and extractum carnis or/and corn steep liquor or/and peptone etc.;

Said inorganic salt be selected from phosphoric acid salt or/and manganese salt or/and sylvite etc.; Said phosphoric acid salt such as SODIUM PHOSPHATE, MONOBASIC or/and Sodium phosphate dibasic etc.; Said manganese salt such as Manganous chloride tetrahydrate or/and manganous sulfate etc.; Said sylvite such as Repone K or/and potassium primary phosphate or/and vitriolate of tartar or/and salt of wormwood or/and saltpetre etc.;

Said VITAMIN is selected from VB or/and VB6 etc.

Its cultural method all is described on many documents, does not repeat them here.Obtain fermented liquid through centrifugal removal thalline with the aforesaid method cultivation, can directly use, or further handle as use ammonium sulfate precipitation, immobilization etc. are to improve access times.Because the enzyme liquor ratio of gained is purer, generally need not purification process.

Said rhodotorula glutinis in the substratum of suitable carbon source, nitrogenous source, inorganic salt, was cultivated 48-96 hour for 22-28 ℃, generally in 24-26 ℃ of cultivation 60-84 hour, centrifugal removal thalline, enzyme liquid is preserved standby in 4 ℃ of refrigerators.

The enzyme liquid of gained is joined the RNA solution that concentration is 5-50mg/mL, and optimum concn is the RNA solution of 10-30mg/mL, and enzyme liquid addition is the 5-30%RNA liquor capacity, and the best is the 10-20%RNA liquor capacity.In 30-80 ℃, best 40-70 ℃, reacted 1-4 hour, can obtain 3 '-mixed nucleotide solution, the classical separation method of utilization can obtain high-purity single Nucleotide.

Analytical procedure:

The vitality test of 3 '-phosphodiesterase is referring to document " Sun Rongrong chemical reagent, 1983,5 (2): 103-106 ".

3 '-Nucleotide adopts HPLC to analyze, and actual conditions is as follows:

HPLC: Tianjin, island; The UV detector;

Post: Hypersil ODS (4.6 * 250mm)

Moving phase: 50mmol/L potassium primary phosphate-dipotassium hydrogen phosphate (pH5.0)

Flow velocity: 1mL/min

Column temperature: 30 ℃

The said method of the present invention can obtain the enzyme liquid of higher vigor, and general vigor is at the 250U/mL fermented liquid; It almost is a times of bibliographical information; Gained enzyme liquid can be placed 1.5 hours and keeps the wherein vigor more than 70% at 70 ℃; The enzyme liquid of gained need not purifying, can be directly used in enzyme reaction, and the RNA degradation rate reaches more than 90%, is mainly four kinds of 3 '-Nucleotide in HPLC analytical reaction liquid.Use method of the present invention to improve production efficiency greatly, operate very simple, the method that a kind of height is economical and practical.

Description of drawings

Fig. 1 is the thermostability of 3 '-phosphodiesterase.

Fig. 2 is the HPLC analysis chart of enzymolysis solution.

Embodiment

Embodiment 1

Rhodotorula glutinis CICC1691 is scraped next ring from the inclined-plane, be inoculated in aseptic culture medium I, in 26 ℃ of shaking culture 36 hours, centrifugal collecting cell, and with stroke-physiological saline solution washing three times, resuspending is in stroke-physiological saline solution, and to adjust cell concn be 10 ⁸About individual/mL, the uviolizing some time, coat dull and stereotyped go up (medium ii), cultivated 48 hours for 26 ℃, get the same position that single bacterium colony point is connected to two flat boards, branch is arranged in 26 ℃ and 30 ℃ and cultivated two days.Select in 26 ℃ of growths and 30 ℃ long or growing way is relatively poor single bacterium colony.Through further identifying (fermentation medium ii I), obtain a plant mutant strain QOX 3318 at last.The results are shown in Table 1.

Table 1

Bacterial classification	Temperature (48 hours)		3 '-phosphodiesterase (U/mL)
				26℃	38℃
	CICC 1691	+				+	100
QOX3318			+	-	256

*+grow fine;-do not grow

Substratum I: wort (5 ° of Be), pH=7.0;

Medium ii: add 2% agar in the substratum;

Medium ii I:5% glucose, 0.5% peptone, 0.5% extractum carnis, 0.2% yeast extract paste, 0.05% magnesium sulfate heptahydrate, 0.01%CaCl ₂2H ₂O, 0.2%KNO ₃, pH=7.0

Embodiment 2

QOX 3318 is inoculated in substratum 1 (30mL/250mL triangular flask), cultivated 24 hours for 26 ℃, transfer in the 50L fermentor tank again, inoculum size was cultivated 72 hours for 5%, 26 ℃, and centrifugal removal thalline is collected fermenting enzyme liquid.Getting enzyme liquid lives in 70 ℃ of heating and timing sampling mensuration enzyme.Contrast with CICC1691 simultaneously.The results are shown in Figure 1.Original vigor is: 251U/mL (QOX 3318); 102U/mL (CICC 1691).

Embodiment 3

The enzyme liquid 100mL that gets 3318 fermentations of QOX among the embodiment 2 joins 900mL RNA concentration and is respectively among 5mg/mL, 10mg/mL, 20mg/mL, the 30mg/mL, with 100mol/L acetate-sodium acetate control pH is 4.1,60 ℃ were reacted 2 hours, sampling HPLC analyzes the wherein content of various 3 '-Nucleotide, the results are shown in Table 2 and Fig. 2.Main peak is followed successively by 3 '-CMP, 3 '-UMP, 3 '-GMP and 3 '-AMP among Fig. 2.

Table 2

RNA (mg/mL)	Degradation rate (%)	3 '-nucleotide concentration (mg/mL)
						3’-CMP	3’-AMP	3’-UMP	3’-GMP
		5	92.3	0.65	1.05					0.9	1.2
10	90.5					1.4	2.3	2.1	2.5
		20	90.0	2.6	4.5					4.0	4.9
30	88.7					4.0	6.1	5.9	7.0

Claims (3)

1. a rhodotorula glutinis (Rhodotorula glutinis) QOX 3318 CGMCCNo.1272.

2. a method for preparing 3 '-Nucleotide is characterized in that, comprises that first fermentation culture adopts rhodotorula glutinis (Rhodotorula glutinis) QOX 3318 CGMCC No.1272;

The enzyme liquid of gained is joined the RNA solution that concentration is 5-50mg/mL, and enzyme liquid addition is the 5-30%RNA liquor capacity, in 30-80 ℃, reacts 1-4 hour, promptly obtains 3 '-mixed nucleotide solution, and the classical separation method of utilization can obtain single Nucleotide.

3. method according to claim 2 is characterized in that, the enzyme liquid of gained is joined the RNA solution that concentration is 10-30mg/mL, and enzyme liquid addition is the 10-20%RNA liquor capacity, and temperature of reaction is 40-70 ℃.

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