CN101492649B - Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine - Google Patents

Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine Download PDF

Info

Publication number
CN101492649B
CN101492649B CN2009100252707A CN200910025270A CN101492649B CN 101492649 B CN101492649 B CN 101492649B CN 2009100252707 A CN2009100252707 A CN 2009100252707A CN 200910025270 A CN200910025270 A CN 200910025270A CN 101492649 B CN101492649 B CN 101492649B
Authority
CN
China
Prior art keywords
polylysine
epsilon
arthrobacter globiformis
bacterial strain
arthrobacter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN2009100252707A
Other languages
Chinese (zh)
Other versions
CN101492649A (en
Inventor
齐斌
郑丽雪
陈浩
王立梅
梅艳珍
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Changshu Institute of Technology
Original Assignee
Changshu Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Changshu Institute of Technology filed Critical Changshu Institute of Technology
Priority to CN2009100252707A priority Critical patent/CN101492649B/en
Publication of CN101492649A publication Critical patent/CN101492649A/en
Application granted granted Critical
Publication of CN101492649B publication Critical patent/CN101492649B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a Arthrobacter globiformis QC18 producing epsilon-polylysine. The invention also relates to the application of the strain in producing epsilon-polylysine by fermentation. The Arthrobacter globiformis QC18 has higher capacity for producing epsilon-polylysine, better thermal stability and substrate survivability; and the characteristics of simple operation, stability and good repetitiveness of the Arthrobacter globiformis QC18 applied on the Epsilon-polylysine are used to realize stable and low-cost production of the epsilon-polylysine, thus being beneficial to scale production.

Description

One strain Arthrobacter globiformis bacterial strain and the application in fermentation production of epsilon-polylysine thereof
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of Arthrobacter globiformis Arthrobacter globigormis QC18 that produces epsilon-polylysine, the invention still further relates to the application of this bacterial strain in fermentation production of epsilon-polylysine.
Background technology
(ε-PL) is that a kind of has a broad antifungal spectrum, germ resistance are strong to epsilon-polylysine, the new type natural sanitas of safety non-toxic, have resisting gram-positive bacteria, Gram-negative bacteria, fungi and virus activity and thermostability height, good water solubility, can biological explain, performances such as edible safety have superior antiseptic property.The sanitas kind of Shi Yonging is a lot of in the market, mainly is the synthetic sanitas, and these sanitass are subjected to the influence of pH very big, only just effective under acidic conditions, as Sorbic Acid and sylvite thereof, benzoic acid and sodium benzoate, these Chemical Preservatives have certain toxic side effect to human body.In addition, in fields such as the preparation of gene therapy, microcapsule medicament, macromolecular material, epsilon-polylysine also has purposes widely.Chinese patent 200710067250.7 discloses a strain streptomyces albus and has produced epsilon-polylysine; Chinese patent 200710057098.4 discloses a strain streptomyces albus and has produced epsilon-polylysine; Chinese patent 200510037774.2 discloses a strain backlands spore bacterium.Epsilon-polylysine is the safety food preservation agent in October, 2003 by the FDA approval; 1977, Shima and Sakai isolated the Streptomyces albulus 346 that can produce epsilon-polylysine from soil.Up to now, the research of epsilon-polylysine has particularly realized industrialization in Japan abroad, but this technology also is in laboratory stage in China, and the principal element that restricts its development is to lack the bacterial strain that proterties is stablized the high yield epsilon-polylysine.
Summary of the invention
Technical purpose of the present invention provides a strain and has higher product epsilon-polylysine ability, the new bacterial strain of microorganism of thermostability, substrate tolerance preferably, and the application of this bacterial strain in fermentation production of epsilon-polylysine is provided.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
One, the present invention's screening and separating from soil obtains a strain Arthrobacter globiformis bacterial strain, classification called after Arthrobacterglobiformis QC18, preservation date is on January 8th, 2009, depositary institution's full name is Chinese typical culture collection center, and its preservation registration number is CCTCC NO:M 209015.
(1) screening of bacterial strain and authentication method:
1, primary dcreening operation:
Get natural soil sample of 1g and 10mL sterilized water and be added to 50 ℃ of vibration 1h in the 100mL triangular flask, be diluted to 10 -3Doubly.To get 10 after the dilution of bacterium liquid -2, 10 -3Three extent of dilution bacterium liquid are applied to the primary dcreening operation substratum, and 30 ℃ just have when cultivating the 3rd day bacterium colony to produce than the obvious transparent circle, and the tangible bacterium colony of transparent circle is used as next step multiple sieve.
2, multiple sieve:
The primary dcreening operation bacterial classification inoculation is sieved in the substratum again in 100mL, under 30 ℃, 200rpm condition, cultivate, get the fermented liquid 1mL under 10000r/min centrifugal 10 minutes that cultivates 21h, 48h respectively; Get supernatant liquor 250 μ L and be added in 96 orifice plates, add one of Dragendorff reagent, leave standstill 3～5min behind the mixing and observe.After the hydrolysis of the centrifugal gained supernatant of fermented liquid strong acid, detect through thin layer chromatography analysis, obtain producing the higher bacterial strain of polylysine, bacterial strain called after QC18.
3, the evaluation of bacterial strain:
With QC18 through the Physiology and biochemistry experimental identification, the result shows that QC18 is close with Arthrobacter Arthrobacter., and the 16SrDNA sequential analysis is identified and to be shown that being higher than 99% bacterial strain with this sequence similarity degree is Arthrobacter and belongs to bacterial strain, be 99.3% wherein with bacterial strain Arthrobacter globiformis Arthrobacter globiformis similarity, therefore this bacterial strain called after Arthrobacter globiformis Arthrobacter globiformis QC18 the most at last.
(2) the present invention identifies the biological characteristic of Arthrobacter globiformis QC18:
1, morphological specificity:
Children age culture cell be irregular shaft-like, enter stationary phase after, thalline becomes sphere; Bacterium colony is creamy white, neat in edge, and smooth surface, aerobic, chemoheterotrophy.
2, physiological and biochemical property:
(1) culture temperature: 20～40 ℃, optimum temperuture is 32 ℃.
(2) gelatine liquefication, starch hydrolysis, milk peptonize and are positive.
(3) cellulose utilization is positive.
(4) H2S generates and is negative.
(5) chitinase produces and is positive.
Two, the application of Arthrobacter globiformis Arthrobacter globiformis QC18 bacterial strain of the present invention in fermentation production of epsilon-polylysine relates generally to and utilizes Arthrobacter globiformis Arthrobacter globiformis QC18 fermentation production of epsilon-polylysine and salt thereof.
Concrete application method:
Arthrobacter globiformis Arthrobacter globiformis QC18 was inoculated in the Gao Shi culture medium culturing after 2～3 days, 25～40 ℃ of cultivations on fermention medium, separation and purification forms and the accumulation epsilon-polylysine from substratum at last.
As long as wherein contain proper amount of carbon source, nitrogenous source, inorganic salt and other nutrient substance, any substratum all can use, and generally needn't limit especially in the above-mentioned fermention medium.As for carbon source, preferentially select sucrose, can select carbohydrate such as sucrose, glucose, lactose, fructose, glycerine etc. for use as the carbon source of fermentation; Nitrogenous source can be selected organic nitrogen source or inorganic nitrogen-sourced for use, as organic nitrogenous source wort, yeast extract paste, peptone, wherein with the yeast extract paste best results, as inorganic nitrogen-sourced available ammonium sulfate, ammonium nitrate, ammonium chloride; Inorganic salt can be selected ferrous sulfate, sal epsom, zinc sulfate, calcium chloride etc.; As buffering salt phosphoric acid potassium dihydrogen, dipotassium hydrogen phosphate, the initial pH of fermented liquid can be nature, and general optional 6.0～9.0; The bacterial classification inoculation amount is generally 5～20%, and more preferably 10～15%; Under 150～300rpm rotating speed stirs, maintain the temperature at 25～40 ℃, fermenting to epsilon-polylysine has a large amount of accumulation, and fermentation time is generally 24～72h, preferred 24～48h.
The application of Arthrobacter globiformis Arthrobacter globiformis QC18 bacterial strain of the present invention in fermentation production of epsilon-polylysine is included in shaking culture under the aerobic conditions, stir culture or other method and cultivates.Culture temperature is 25～40 ℃; The pH of substratum is near neutral (pH7.2), and fermentation middle and later periods pH can reduce, and when pH is reduced to 4, adds alkali and keeps pH 4.The alkali that adds is preferably ammoniacal liquor, but sodium hydroxide, potassium hydroxide or other alkali are all available, and epsilon-polylysine accumulates in substratum after common 1～4 day.Fermented liquid centrifugation thalline filters and obtains clear filtrate.
The fermented liquid that fermentation culture 72h is obtained under 10000rpm centrifugal 10 minutes, separating thallus, filtration obtains clear filtrate, transfer pH to 7.5 then, isolate residue, use IRC-50 (Zeo-karb), IRA-402 (anionite-exchange resin) and XT-1006 (Zeo-karb) to carry out purifying, concentrate by reverse osmosis membrane then, thereby the acquisition epsilon-polylysine, the output of separation and purification formation and accumulation epsilon-polylysine reaches 14.6g/L from substratum at last.The gained crude product is about 5200Da through the Coomassie brilliant blue determining molecular weight.
Beneficial effect of the present invention is:
1, the new bacterial strain Arthrobacter globiformis Arthrobacter globiformis QC18 that screens of the present invention can produce epsilon-polylysine; substrate (sugared concentration) tolerance is preferably arranged; the application operating of Arthrobacter globiformis Arthrobacter globiformis QC18 on epsilon-polylysine is simple, stable, good reproducibility and utilize; realized that epsilon-polylysine produces stablely, inexpensive, helped large-scale production.
2, epsilon-polylysine and the salt thereof that obtains by Arthrobacter globiformis Arthrobacter globiformis QC18 fermentative production of the present invention has following physico-chemical property:
(1) water-soluble, hydrochloric acid is slightly soluble in ethanol, is insoluble to organic solvents such as ether, ethyl acetate;
(2) after the HCl hydrolysis with 6N triketohydrindene hydrate is positive;
(3) with after the HCl hydrolysis of 6N, analyse and the thin-layer chromatography detection with ply of paper, find that its hydrolyzed solution is single amino acid, Methionin shows that this product is the high molecular polymer of Methionin;
(4) by the SDS-PAGE electrophoresis detection, its molecular weight is 5200Da.
The preservation date of microorganism Arthrobacter globiformis Arthrobacter globiformis QC18 of the present invention is on January 8th, 2009, and depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, and its preservation registration number is CCTCC NO:M 209015.
Embodiment
The substratum that uses in the embodiment of the invention:
Primary dcreening operation substratum (g/L): sucrose 20g, yeast extract paste 0.2g, NaH 2PO 41g, KH 2PO 41g, MgSO 47H 2O0.25g, (NH 4) 2SO 42g, methylene blue 0.02g, K 2Cr 2O 70.05g agar 18g is settled to 1L, pH7.0 is in 1 * 10 5The Pa 25min that sterilizes.
Sieve substratum (g/L) again: glycerine 20g, sucrose 20g, yeast extract paste 3g, NaH 2PO 41g, KH 2PO 41g, MgSO47H 2O 0.25g, (NH 4) 2SO 45g is settled to 1L, pH7.0,1 * 10 5The pa 25min that sterilizes.
Fermention medium (g/L): sucrose 20g, yeast extract paste 3g, NaH 2PO 41g, KH 2PO 41g, MgSO 47H 2O0.25g, (NH 4) 2SO 45g is settled to 1L, and pH 7.0, in 1 * 10 5The pa 25min that sterilizes.
Gao Shi substratum (g/L): MgSO 47H 2O 0.5g, FeSO 40.01g, KNO 31g, NaC1 0.5g, agar 20g, K 2HPO 40.5g, Zulkovsky starch 20g, pH 7.2～7.4, and 1 * 10 5The Pa 25min that sterilizes.
Embodiment 1
Screening, the qualification program of present embodiment explanation Arthrobacter globiformis Arthrobacter globiformis QC18.
1, primary dcreening operation:
Soil sample and the 10mL sterilized water of getting (Changshu Yu Shan, ecological garden etc.) that 1g selects at random are added to 50 ℃ of vibration 1h in the 100mL triangular flask, are diluted to 10 -3Doubly.To get 10 after the dilution of bacterium liquid -2, 10 -3Three extent of dilution bacterium liquid are applied to the primary dcreening operation substratum, and 30 ℃ just have when cultivating the 3rd day bacterium colony to produce than the obvious transparent circle, and the tangible bacterium colony of transparent circle is used as next step multiple sieve.
2, multiple sieve:
The primary dcreening operation bacterial classification inoculation is sieved substratum again in 100mL,, cultivate under the 200rpm condition, get the fermented liquid 1mL under 10000r/min centrifugal 10 minutes that cultivates 21h, 48h respectively at 30 ℃; Get supernatant liquor 250 μ L and be added in 96 orifice plates, add one of Dragendorff reagent, leave standstill 3～5min behind the mixing and observe.The centrifugal gained supernatant of fermented liquid behind the usefulness 6mol/L hydrochloric acid soln hydrolysis 12h, is detected through thin layer chromatography analysis in peace is fallen bottle, and thin layer plate adopts silica-gel plate, and developping agent is a propyl carbinol: acetate: water=4: 1: 1 (volume ratio), L-Methionin is as standard substance.With the most tangible strain bacterium called after QC18 of Methionin colour developing spot in the analytical results.
3, the evaluation of bacterial strain:
With QC18 through the Physiology and biochemistry experimental identification, the result shows that QC18 is close with Arthrobacter Arthrobacter., and the 16SrDNA sequential analysis is identified and to be shown that being higher than 99% bacterial strain with this sequence similarity degree is Arthrobacter and belongs to bacterial strain, wherein belonging to similarity with strains A rthrobacter globiformis is 99.3%, therefore this bacterial strain called after Arthrobacter globiformis QC18 the most at last.
Embodiment 2
The biological characteristic of present embodiment explanation Arthrobacter globiformis QC18.
1, morphological specificity:
Mycelial growth is good, and aerial hyphae forms long spore chain, sphere, and size is more even, is the sporophore that chain generates aerial hyphae.
2, physiological and biochemical property:
(1) culture temperature: 20～40 ℃, optimum temperuture is 32 ℃.
(2) gelatine liquefication, starch hydrolysis, milk peptonize and are positive.
(3) cellulose utilization is negative.
(4) H 2S generates and is negative.
(5) chitinase produces and is negative.
Embodiment 3
The content assaying method of present embodiment explanation epsilon-polylysine.
Epsilon-polylysine assay: adopt ultraviolet-visible spectrophotometry, with reference to Itzhaki method of the prior art.
The epsilon-polylysine standard curve making:
Accurately draw the polylysine reference liquid of 2.0g/L respectively, 0,20,40,60,80,100 μ L are in centrifuge tube, add distilled water to 100 μ L, add 0.1mmol/L sodium phosphate buffer 1.9mL and 0.1mmol/L and the methyl orange solution 2mL of class then successively, with carrying out thermal agitation 30min behind the silicon plug jam-pack immediately, fully after the reaction, in the centrifugal 10min of 4000rpm, get 20 times of supernatant liquor dilutions, the 465nm place surveys its OD value.The equation of linear regression that is obtained by the match of minimum secondary phase multiplication is y=2.90047-4.8752x, r 2=0.9995, wherein y represents epsilon-polylysine concentration, and x represents light absorption value, r 2Represent relation conefficient.
Embodiment 4
The separation purification method of present embodiment explanation epsilon-polylysine.
The fermented liquid that fermentation culture 72h is obtained under 10000rpm centrifugal 10 minutes, separating thallus, filtration obtains clear filtrate, transfer pH to 7.5 then, isolate residue, use IRC-50 (Zeo-karb), IRA-402 (anionite-exchange resin) and XT-1006 (Zeo-karb) to carry out purifying, concentrate by reverse osmosis membrane then, thereby the acquisition epsilon-polylysine, the output of separation and purification formation and accumulation epsilon-polylysine reaches 14.6g/L from substratum at last.The gained crude product is about 5200Da through the Coomassie brilliant blue determining molecular weight.
Embodiment 5
The application of present embodiment explanation Arthrobacter globiformis Arthrobacter globiformis QC18 bacterial strain in fermentation production of epsilon-polylysine.
Arthrobacter globiformis Arthrobacter globiformis QC18 is inoculated in Gao Shi to be cultivated based on 30 ℃ of cultivations.Cultivate after 2～3 days, on fermention medium 30 ℃, 200rpm cultivates, the fermented liquid that fermentation culture 120h is got under 10000rpm centrifugal 10 minutes, separating thallus filters and obtains clear filtrate, transfer pH to 7.5 then, embodiment 4 described method purifying, thus epsilon-polylysine obtained, and the output of separation and purification formation and accumulation epsilon-polylysine reaches 21.3g/L from substratum at last.

Claims (2)

1. a strain Arthrobacter globiformis (Arthrobacter globiformis) bacterial strain, classification called after Arthrobacter globiformis (Arthrobacter globiformis) QC18, preservation date is on January 8th, 2009, depositary institution's full name is Chinese typical culture collection center, and its preservation registration number is CCTCC NO:M209015.
2. the application of Arthrobacter globiformis Arthrobacter globiformis QC18 according to claim 1 in fermentation production of epsilon-polylysine.
CN2009100252707A 2009-03-05 2009-03-05 Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine Expired - Fee Related CN101492649B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2009100252707A CN101492649B (en) 2009-03-05 2009-03-05 Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2009100252707A CN101492649B (en) 2009-03-05 2009-03-05 Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine

Publications (2)

Publication Number Publication Date
CN101492649A CN101492649A (en) 2009-07-29
CN101492649B true CN101492649B (en) 2010-12-29

Family

ID=40923441

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2009100252707A Expired - Fee Related CN101492649B (en) 2009-03-05 2009-03-05 Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine

Country Status (1)

Country Link
CN (1) CN101492649B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483813A (en) * 2003-02-14 2004-03-24 中国科学院南京土壤研究所 Heterotrophic nitrobacteri, culturing method and application thereof
CN101250576A (en) * 2008-04-01 2008-08-27 天津科技大学 Method for producing 17 alpha-methyl-teslosterone by employing spherical arthrobacterium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1483813A (en) * 2003-02-14 2004-03-24 中国科学院南京土壤研究所 Heterotrophic nitrobacteri, culturing method and application thereof
CN101250576A (en) * 2008-04-01 2008-08-27 天津科技大学 Method for producing 17 alpha-methyl-teslosterone by employing spherical arthrobacterium

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
宋玉栋等.嗜热溶胞土芽孢杆菌(Geobacillus sp.)SY-9 的基本特性.《中国环境科学》.2007,第27卷(第4期),456-460. *
魏杰等.一株高产谷氨酰胺合成酶的LNU 0165菌的鉴定.《辽宁大学学报自然科学版》.2006,第33卷(第4期),349-352. *

Also Published As

Publication number Publication date
CN101492649A (en) 2009-07-29

Similar Documents

Publication Publication Date Title
CN103865855B (en) Bacillus subtilis strain and its application
CN101792727B (en) Bacillus coagulans and application thereof in L-sodium lactate preparation
CN101914478B (en) Bacillus subtilis and application thereof
CN103305430B (en) Laccase generation cerrena and application thereof
CN105017086A (en) Separation and purification method for L-citrulline
CN105420140A (en) Bacillus amyloliquefaciens and application thereof
CN106278493A (en) The classification enzymatic isolation method preparation method containing oligosaccharide seaweed organic fertilizer
CN102250808A (en) Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof
CN110042072A (en) A kind of aflatoxin degradation B1Bacterial strain and its application
CN104531583A (en) Pseudomonas and microbial flocculant formed by fermenting pseudomonas
CN103627698A (en) Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain
CN102994430A (en) Bacterial cellulose production strain and application thereof
CN106987540B (en) One plant of Rhodobacter capsulatus and its application
CN103374537A (en) Method for preparing enduracidin and strain produced thereby
CN105176859A (en) Strain MQO-153 for production of arginine deiminase
CN105175275B (en) A kind of isolation and purification method of L ornithine
CN102286402B (en) Soybean rhizosphere azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on various pathogenic fungi and purpose thereof
CN103305481B (en) Method for producing laccase by fermenting cerrena unicolor
CN103194500A (en) Fermentation preparation method of epsilon-poly-L-lysine
CN101492649B (en) Spherical node bacillus bacterial strain and uses in fermentation production of Epsilon-polylysine
CN102321555B (en) Endophyte producing termite-killing/resistant compound
CN100390295C (en) Microorganism polysaccharide and its preparation method and application
CN102220404A (en) Preparation method of compound microbial flocculant for cyanobacterial bloom
CN105177075A (en) Method for preparation of L-citrulline with arginine as raw material
CN101892163B (en) Penicillium camemberti and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20101229

Termination date: 20200305

CF01 Termination of patent right due to non-payment of annual fee