CN102433288A - Strain for producing ornithine and method for biologically synthesizing ornithine with same - Google Patents

Abstract

The invention provides a strain for producing ornithine and a method for biologically synthesizing ornithine with the same, belonging to the food biotechnology field. The strain and the method have the following beneficial effects: the invention relates to rummeliibacillus pycnus (SK23-002) screened from the soil; the collection number of rummeliibacillus pycnus is CCTCC NO:M2011466; the sporeformer is taken as the fermentation strain, glucose is taken as the carbon source and yeast cream and peptone are taken as nitrogen sources to form a fermentation medium to ferment ornithine or a thallus containing arginase, and the thallus is added to 5-20% of arginine solution to synthesize ornithine through transformation; through detection, the transformation rate can be more than 80%; the fermentation process can be independent of arginine induction; ornithine produced by the method is safe and reliable, is a functional product with great market potential and is widely applied to such industries as food, cosmetics, medicines and the like; and the method can be used for efficiently producing ornithine and is suitable for large scale production.

Description

A kind of bacterial strain that produces ornithine and with the method for this bacterial strain biosynthesizing ornithine

Technical field

The thalline that the present invention relates to produce a kind of novel microorganism bacterial strain and the cultivation and fermentation production ornithine thereof of ornithine or contain arginase; And this thalline is used for the biological method for preparing ornithine; Belong to technical field of food biotechnology, be widely used in industries such as food, makeup, medicine.The present invention relates to derive from soil in particular Rummeliibacillus pycnus(SK23.002), deposit number is CCTCC NO:M 2011466, utilizes this bacterial strain biosynthesizing ornithine (ornithine).

Background technology

In recent years find that ornithine can stimulate pituitary secretion growth hormone; Promote the metabolism (basal metabolism) of protein synthesis and sugar and fat; The ornithine of under the combination motion conditions, ingesting can reduce body fat and pile up; Strengthen muscle and muscle power play the effect of Weight-reducing health, and ornithine also can promote enteron aisle to grow up and improve the intestine immunity function in addition; The L-ornithine also is that the synthetic body before of cell growth factor polyamine has the skin makeup of helping effect, on raising immunologic function and anti-cancer function, certain effect is arranged also.The L-ornithine is added in sportsmen's healthcare products branched-chain amino acid, also has the debitterize effect, so ornithine is a multi-functional amino acid, so become the focus of concern.

The made ornithine of the present invention has following characteristic.Molecular formula: C ₅H ₁₂N ₂O ₂, molecular weight: 132.19, fusing point: 140 ℃ (grain, 120 ℃ are softening), and solvability: soluble in water and ethanol, be slightly soluble in ether, its solution is alkalescence, and soluble in water, ethanol is slightly soluble in ether.What usually use as reagent is its single salt, and L-ornithine list salt is soluble in water, is insoluble to methyl alcohol, ethanol, ether.In vivo, ornithine is mainly participated in uric acid circulation, plays an important role for the discharge of ammonia-state nitrogen in the body.Ornithine is used for the tired fizz of configuration restore with l-arginine usually pharmaceutically except that as reagent and injection liquid.In recent years, the application of ornithine on foodstuffs industry and medicine industry is increasingly extensive.

The production of ornithine at present mainly contains synthesis method, enzyme process and 3 kinds of methods of fermentation method.Synthesis method is the preparation method when studying ornithine in early days, and its process is complicated, and cost is higher, and D-ornithine and L-ornithine have, and bad separation is so be eliminated.Enzyme process be l-arginine under the effect of arginase, generate the L-ornithine, this method product purity is high, but costs an arm and a leg, l-arginine and arginase all are difficult for obtaining, and are limited to prepared in laboratory.The ornithine of fermentative Production, this method is simple and easy to do, as long as select suitable bacterial classification, can reach higher output, is applicable to commercial scale prodn.So,, the research of Production by Microorganism Fermentation ornithine is just had important in theory meaning and economic implications along with the application of ornithine on foodstuffs industry and medicine industry is increasingly extensive.

The inventor investigates and has studied prior art further; And the bacterial strain that from soil, sifts out many strains production ornithines is studied; Finishing screen is chosen the new microbe of strain ability high yield ornithine; Can reach the transformation efficiency more than 90% after the optimization, and obtain purer ornithine through means such as separation and purification and concentrate dryings.Based on above-mentioned discovery the present invention has been proposed.

Summary of the invention

The purpose of this invention is to provide a kind of new mikrobe, it can the high yield ornithine, and can not rely on arginic inducing in the fermenting process.

Its two, provide the method that a kind of this bacterial strain is produced ornithine, to obtain high transformation efficiency.

Moreover, provide a kind of ornithine separation and purification and purified method, to obtain highly purified ornithine.

To achieve these goals, the present invention provides a kind of soil of deriving from Rummeliibacillus pycnus(SK23-002), deposit number is CCTCC NO:M 2010073.

Technical scheme of the present invention: the bacterial strain of ornithine, its called after of classifying are produced in a strain Rummeliibacillus pycnus(SK23-002), be to draw through learning character, the analysis of biochemical property and the mensuration of 16sRNA sequence according to the mushroom of certain rule.Utilize this mikrobe, the method for fermentative prodn ornithine, step is following:

(1) seed culture

Seed culture medium: L-Arg 1-20g/L, yeast extract paste 1-20g/L, peptone 1-20g/L, glucose 5-100g/L, inorganic nitrogen-sourced 1.0-20.0g/L and add or do not add small amounts of inorganic salt etc. transfers pH6.0-7.5;

The seed culture condition: CCTCC NO:M 2011466 bacterial strains are cultivated this bacterial strain of 10-15h activation in seed culture medium under 30-37 ℃, the hunting speed of 150-250rpm.

(2) fermentation culture

Fermention medium: yeast extract paste 1-20g/L, peptone 1-20g/L, glucose 1-100g/L, L-Arg 1-20g/L, inorganic nitrogen-sourced 1.0-20.0g/L and add or do not add small amounts of inorganic salt etc. transfers pH6.0-7.5;

5-72h ferments in fermention medium under fermentation condition: the inoculum size 1%-10%, 30-37 ℃, the condition of rotating speed 100-500rpm.

(3) fermentation aftertreatment

Fermented liquid is collected fermented liquid that contains ornithine and the wet thallus that contains arginase behind frozen centrifugation; And clean thalline with the 0.1mol/L acetate buffer of 0.1% saline water and pH6.0; Get resting cell and carry out full cell transformation reaction, through detecting transformation efficiency more than 80%.

(4) full cell transformation reaction: substrate l-arginine concentration 10-200g/L, resting cell amount 0.1-10g/L, this is reflected at pH4.0-8.0, conversion reaction time 5-40h under 30-70 ℃ the condition, the reaction solution after obtaining transforming,

(5) separation and purification of ornithine

To contain the fermented liquid of ornithine or the reaction solution after the conversion and after decolouring, utilize strongly acidic cationic exchange resin 001 * 7, the ammoniacal liquor wash-out carries out the ornithine separation and purification.

Beneficial effect of the present invention: the present invention relates to that a strain is screened and come from soil Rummeliibacillus pycnus(SK23-002), deposit number is CCTCC NO:M 2011466, with this sporeformer for going out fermentation strain; With glucose is that carbon source, yeast extract paste and peptone are composition fermention mediums such as nitrogenous source; Fermentative prodn contains the thalline of arginase, and fermentation gained thalline adds the transfer of 5%-20% arginine solution to and is combined to ornithine, can know through detecting; Transformation efficiency can reach more than 80%, and this fermenting process can not rely on the l-arginine inducing action.The ornithine that the inventive method is produced is safe and reliable, is a kind of functional product that market potential is arranged very much, is widely used in industries such as food, makeup, medicine.This invention can be produced ornithine efficiently, is suitable for carrying out scale operation.

The biological material specimens preservation: a kind of bacterial strain that produces ornithine, its classification called after ( Rummeliibacillus pycnus) SK23.002, be preserved in Chinese typical culture collection center, be called for short CCTCC, address: Chinese Wuhan Wuhan University, deposit number is CCTCC NO:M 2011466, preservation date on December 12nd, 2011.

Embodiment

Below be Rummeliibacillus pycnus(SK23.002) carry out the embodiment of fermentative prodn ornithine, but technical scope of the present invention is not limited to listed several instances, under the prerequisite that does not change its main points, can make various changes and implement.In addition, technical scope of the present invention prolongs and impartial scope.

The fermentation culture of embodiment 1 SK23.002 bacterial strain

Use bacterial strain SK23.002, containing L-Arg 0.5%, glucose 1%, yeast extract paste 0.5%, peptone 0.5%; Inorganic nitrogen-sourced 0.1% and contain or do not contain small amounts of inorganic salt etc.; Transfer in the substratum of pH6.0,37 ℃, cultivate 22h under the condition of 190 rpm; Frozen centrifugation makes the resting cell with arginase work.

Embodiment 2 conversion reactions

Use embodiment 1 gained thalline, in containing the 100mM acetate buffer solution (HAc-NaAc, pH6.0) that the l-arginine mass concentration is 50g/L, resting cell amount 0.1-10g/L, under 37 ℃, carry out conversion reaction 5-40h, synthetic ornithine.

The mensuration of embodiment 3 ornithine output

The reaction solution that carries out among the embodiment 2 through the enzyme that goes out (TCA deposition), dilution, through the Agilent liquid chromatograph, is carried out quantitatively l-arginine, ornithine respectively.Analysis condition is following: instrument model: Agilent 1100, chromatographic column: (250*4.6) mm 5 μ m ODS HYPERSIL, column temperature: 40 ℃.

The evaluation that embodiment 4 novel ornithines produce bacterium

Novel ornithine is produced bacterium be sent to morphological specificity, physio-biochemical characteristics and the 16SrRNA gene sequencing that this bacterium is measured at Chinese typical culture collection center, be accredited as Rummeliibacillus pycnus,It is safe bacterial strain.

The separation and purification of embodiment 5 ornithines

The reaction solution that transforms gained after decolouring, is utilized strongly acidic cationic exchange resin 001 * 7, carry out wash-out, obtain the ornithine of purifying with the ammoniacal liquor of different concns.

Claims (2)

1. the bacterial strain of ornithine is produced in a strain, its classification called after ( Rummeliibacillus pycnus) SK23.002, being preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M 2011466.

2. the method for producing ornithine with described CCTCC NO:M 2011466 strain fermentations of claim 1; It is characterized in that; Through fermentative prodn ornithine and have the active somatic cells of arginase, carry out full cell transformation reaction then, synthetic ornithine through fermentative prodn; Step is:

(1) seed culture

Seed culture medium: L-Arg 1-20g/L, yeast extract paste 1-20g/L, peptone 1-20g/L, glucose 5-100g/L, inorganic nitrogen-sourced 1.0-20.0g/L transfers pH6.0-7.5, the deionized water preparation;

The seed culture condition: CCTCC NO:M 2011466 bacterial strains are cultivated this bacterial strain of 10-15h activation in seed culture medium under 30-37 ℃, the hunting speed of 150-250rpm;

(2) fermentation culture

Fermention medium: yeast extract paste 1-20g/L, peptone 1-20g/L, glucose 1-100g/L, L-Arg 1-20g/L, inorganic nitrogen-sourced 1.0-20.0g/L transfers pH6.0-7.5, the deionized water preparation;

5-72h ferments in fermention medium under fermentation condition: the inoculum size 1%-10%, 30-37 ℃, the condition of rotating speed 100-500rpm;

(3) fermentation aftertreatment

Fermented liquid is collected fermented liquid that contains ornithine and the wet thallus that contains arginase behind frozen centrifugation; And clean thalline with the 0.1mol/L acetate buffer of 0.1% saline water and pH6.0; Get resting cell and carry out full cell transformation reaction, through detecting transformation efficiency more than 80%;

(4) full cell transformation reaction: substrate l-arginine concentration 10-200g/L, resting cell amount 0.1-10g/L, this is reflected at pH4.0-8.0, conversion reaction time 5-40h under 30-70 ℃ the condition, the reaction solution after obtaining transforming,

(5) separation and purification of ornithine

To contain the fermented liquid of ornithine or the reaction solution after the conversion and after decolouring, utilize strongly acidic cationic exchange resin 001 * 7, the ammoniacal liquor wash-out carries out the ornithine separation and purification.