CN1170925C - Fermentation production process of alkaline mycose lyase and microbe for producing the lyase - Google Patents
Fermentation production process of alkaline mycose lyase and microbe for producing the lyase Download PDFInfo
- Publication number
- CN1170925C CN1170925C CNB001211064A CN00121106A CN1170925C CN 1170925 C CN1170925 C CN 1170925C CN B001211064 A CNB001211064 A CN B001211064A CN 00121106 A CN00121106 A CN 00121106A CN 1170925 C CN1170925 C CN 1170925C
- Authority
- CN
- China
- Prior art keywords
- lyase
- gram
- culture
- grams
- alkaline
- 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
Links
Abstract
The present invention provides novel alkalimonas amylolytica N19-2 and a microorganism fermentation method by the alkalimonas amylolytica N19-2 to produce alkaline algal polysaccharides lyase. The novel alkalimonas amylolytica N19-2 used in the present invention takes sodium alginate, peptone, etc. as raw materials to produce the alkaline algal polysaccharides lyase by fermentation. The present invention has a simple production method, the enzyme activity of the fermentation liquid reaches more than 100 u/ml, and the extract absorptivity reaches more than 80%. The pH value of the enzyme for the optimal reaction is 9.5, the temperature for the optimal reaction is 55 DEG C, and the enzyme has important application value on producing alga oligosaccharide.
Description
Technical field
The present invention relates to a kind of new microorganism and utilize this microbial fermentation to produce the method for enzyme.
Background technology
Sargassum polysaccharides lyase or alginate lyase (Alginate lyase.EC4.2.2.3.), by β-elimination mechanism Sargassum polysaccharides is degraded to unsaturated uronic acid oligomer-Lalgine oligosaccharides (alginicacid oilgosaccharides) and unsaturated uronic acid monomer (uronic acid), according to the effect of enzyme to substrate, it is polyM lyase and polyG lyase that this enzyme is divided into two types.
Since the nineties, the using value of alginate lyase in the Bio-engineering Products research and development causes concern, and it not only can develop new physiologically active substance, the more important thing is and has opened up a new industrial application.Except directly being applied to treat the pneumonia that is caused by the pathogenic bacterium Pseudomonas aeruginosa as the medicine enzyme, the important directions that the Sargassum polysaccharides lyase is used is the Production by Enzymes alga oligosaccharide, for the exploitation of new drug, novel functional food product provides new tool.The physiological function of the enzymolysis product that particularly is called as oligosaccharide (oligosaccharins)---alga oligosaccharide is constantly revealed, has caused the interest that people are bigger.The physiological function of alga oligosaccharide comprises the propagation that promotes plant-growth, stimulation of bifidobacteria, suppresses tumour, brings high blood pressure down, activates people's epidermal keratinocyte etc.Englishman in 1997 finds that again alga oligosaccharide can make penicillin G output increase 50-150% (Ariyo B.T.et al., Biotechnol.Bioeng.53:17-20,1997).
The Sargassum polysaccharides lyase is carried out big quantity research, comprised this enzyme of marine microorganism and Lu Sheng microorganisms, related to the each side such as generation, purifying, genetic analysis of this enzyme.The main reference document comprises Marcello, A.et al.:FEMS Microbiol.Lett.; 136 (1): 39-44,1996; Aasen I.M.et al, 37:55-60,1992; Ostgarol K.et al.:EnzymeMicrobiol.Technol.1993,15:756-763; Kinoshita, S.et al.:J.Ferment.Bioeng.72:74-78,1991; Murata, K.et al.:Comments Agric.﹠amp; FoodChemistry, 3 (2): 87-110,1994; Sutherland, Ian W.:Polysaccharide lyases, FEMS Microbiology Reviews, 16:323-347,1995; Sawabe, T.et al.:Carbohydr.Res.; 304 (1): 69-76,1997; Shimokawa T.et al.:Biosci.Biotech.Biochem., 61 (4): 636-640,1997; Malissard M.et al.:FEMS Microbiol.Lett.126 (2): 105-111,1995; Dyrset N.et al.:Appl.Microbiol.Biotechnol.41:523-530,1994; Nakagawa, A.et al.:J.Appl.Microbiol.; 84 (3): 328-335,1998; Ertesvag, H.et al.:J.Bacteriol., 180 (15): 3779-3784,1998; Baron, A.J.et al.:Gene, 143 (1): 61-66,1994; Brown, B.J.et al.:Appl.Environ.Microbiol.57 (6): 1870-1872,1991; Hicks, S.J.et al.:Enzyme Microbiol.Technol., 19 (1): 68-73,1996.
Some effort have been made, attempt the Sargassum polysaccharides lyase is applied to the production of alga oligosaccharide, as JP63039581A2, JP63214192A2, US5516666, US4958016, patents such as EP0979301A1, described lyase is from Altermonas sp., Flavobacterium sp., Bacillus sp., Psudomonas sp. etc., and exploitation is arranged
The characteristic enzymeTrial, as US5139945, WO9002794A1, the high temperature enzyme from Bacillussteraothermophilus of report such as AU4335189A1, but these enzymes still can't satisfy the actual needs that is applied to oligosaccharides production at aspects such as enzyme activity, stability, transformation period at present, existing Sargassum polysaccharides lyase is applied to alga oligosaccharide production and mainly has two problems, and promptly enzyme activity is low and active complicated.Therefore the enzyme of practical application still lacks very much.
Summary of the invention
The object of the present invention is to provide a kind of production bacterial strain of new alkaline mycose lyase and the mass production method of alkaline mycose lyase.
The extreme enzyme of halophile at first can be avoided the interference of glycosidase activity for the development tool Special Significance of Sargassum polysaccharides lyase, overcomes the active complicated defective of general Sargassum polysaccharides lyase; In addition because the molecular characterization of Sargassum polysaccharides, sex change forms irreversible gel under the acidic conditions, and the easy swelling of its molecule under the alkaline condition, also help the cracking of enzyme, and metal ion complex is many to be formed under alkaline condition, and with the difference of pH in the processing process, but the trans-substitution of metal ion phase, the alkaline alginate lyase of therefore having a liking for the alkali bacterium has peculiar advantage.In the lyase of having found, its reaction pH is many between 7-11, but the reaction pH of the Sargassum polysaccharides lyase of having reported generally is neutral, report (the Japanese Patent that has only a facultative Alkaliphilic bacillus Bacillus sp.M-2, special public clear 52-2997), its optimal reaction pH is 9, and yield of enzyme is low, 3u/ml is only arranged, also do not carry out the research of zymotechnique.The Gram-negative bacteria that obligate is had a liking for alkali does not appear in the newspapers as yet.
Bacterial classification involved in the present invention is that obligate is had a liking for the alkali Gram-negative bacteria, and this bacterial classification is not grown below 8 at pH, and the optimal reaction pH of the enzyme of generation is 9.5, and the fermentation broth enzyme vigor is up to more than the 100 units per ml fermented liquids.
Bacterial strain of the present invention is for separating from the salt Zymomonas mobilis N19-2 of coastal area of china (Halomonas sp.N19-2).Bacterial strain N19-2 does not produce brood cell, G
-, the pH scope of growth is pH 8-11.5, the suitableeest growth pH9.5-10, and well-grown in the NaCl of 0.5-8%, the suitableeest is 3%.The cultural characteristic and the physiological and biochemical property of this bacterial strain are listed in the table below.
Table 1, the feature of bacterial strain N19-2
| Cellular morphology brood cell's Gram’s staining flagellum oxidizing ferment catalase Starch Hydrolysis gelatin liquefaction urase casein hydrolysis nitrate reduction indoles produces H2S Vp reaction utilizes carbohydrate to produce sour grapes sugar galactolipin arabinose mannose growth temperature anaerobic growth DNA G+C% | Shaft-like do not produce feminine gender extremely give birth to positive+---++--+--+20-50 ℃, the suitableeest 35 ℃+52% |
This bacterial strain is the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms in Microbe Inst., Chinese Academy of Sciences on June 28th, 2000, the address: carried out preservation No. 13 Institute of Micro-biology in north, BeiJing ZhongGuanCun), preserving number is CGMCC No.0464.
The invention provides a kind of method of producing the Sargassum polysaccharides lyase by microbial fermentation, this method may further comprise the steps:
(1) with salt Zymomonas mobilis N19-2 in seed culture medium, be fit to carry out seed culture under the culture condition of seed growth;
(2) culture that (1) is gathered in the crops under the culture condition that is fit to thalli growth, carries out fermentation culture in containing the fermention medium of sodium alginate;
(3) Shou Huo fermented liquid concentrates by Plate Filtration and Hollow Fiber Ultrafiltration, makes the alkaline mycose lyase.
Seed culture medium of the present invention is to contain starch 5 grams, peptone 10 grams, yeast powder 5 grams, K in every liter of nutrient solution
2HPO
41 gram, MgSO
4-7H
2O 0.15 gram, NaCl 30 grams, Na
2CO
310 grams, fermention medium are to contain Na-alginate 5-20 gram, peptone 4.5 grams, yeast powder 5-7 gram, K in every liter of nutrient solution
2HPO
41-3 gram, MgSO
47H
2O 0.1-0.3 gram, NaCl 10-50 gram, Na
2CO
38-15 gram, soya-bean oil 2-5ml.
In the present invention, the culture condition that is fit to seed growth is 35 ℃ of temperature, 220 rev/mins of shaking speed, shaking culture 24 hours; The fermentation culture conditions of described suitable thalli growth is temperature 32-37 ℃, air flow 1: 0.5-1: 1.0, and rotating speed 200-450 rev/min, fermentation time is 32-48 hour.
In the present invention, the alkaline mycose lyase optimal reaction pH of acquisition is 9.5, and optimal reactive temperature is 55 ℃.
Liquid seed culture medium of the present invention contains (grams per liter): starch 5, peptone 10, yeast powder 5, K
2HPO
41, MgSO
47H
2O 0.15, and NaCl 30, Na
2CO
310.Solid inclined-plane seed culture medium is to add 2% agar powder in the above-mentioned substratum.Fermention medium (grams per liter): sodium alginate 10-18, peptone 4.5, yeast powder 5-7, K
2HPO
41-3, MgSO
47H
2O 0.1-0.3, NaCl10-30, Na
2CO
38-15, soya-bean oil 2-5ml.
Get on the solid slant culture base well-grown culture a little, be inoculated in the 250ml triangular flask that the 50ml seed culture medium is housed, put 35 ℃, cultivate 24 hours on 220 rev/mins the rotary shaker as primary seed solution.Inoculum size with 4% is transferred first order seed in the 3000ml triangular flask that the 1000ml seed culture medium is housed, and above-mentioned condition was cultivated 24 hours, as secondary seed solution, with the inoculum size of 3-5%, secondary seed solution was transferred in seeding tank or fermentor tank again.Seeding tank is 50 liters of jars, stocking volume 20-30 liter, and leavening temperature 32-37 ℃, air flow 1: 0.5-1: 1.0, rotating speed 400-450 rev/min, time 20-28 hour.Fermentor tank is 250 liters of-1 ton of jars, charge amount 120-600 liter.Leavening temperature 32-37 ℃, air flow 1: 0.5-1: 1.0, rotating speed 200-450 rev/min.Fermentation time 32-48 hour.
For the fermented liquid that obtains as stated above, wherein the enzyme activity of alkaline mycose lyase is more than 100u/ml, and the enzyme activity determination method is measured with reference to the method for Horikoshi K..et al.JP877677.Specifically, with 0.9ml, 0.5% (W/V) Sargassum polysaccharides is made substrate, with pH 10,0.05MOL/L glycine-NaOH damping fluid adds 0.1ml dilution enzyme liquid, 55 ℃ are incubated 10 minutes, survey the reducing sugar that produces with edlefsen's reagent, 1 enzyme activity unit is defined as under the above-mentioned reaction conditions, and one minute release 1mMOL is equivalent to the enzyme amount of the reducing sugar of D-seminose.
Fermented liquid concentrates through Plate Filtration, hollow fiber column ultrafilter can obtain purified concentrated enzyme liquid, 10 times of cycles of concentration, and the enzyme yield is more than 85%.
Utilize the new novel alga polysaceharide lyase of having a liking for alkali salt Zymomonas mobilis and method of the present invention production of the present invention, compare with the enzyme that prior art discloses, its enzymic activity height, single-minded, Heat stability is good is used for the cracking Sargassum polysaccharides and has significant advantage.The invention provides obligate and have a liking for Aral Sea polysaccharides lyase generation bacterium, provide feasible technology for realizing alkaline mycose lyase mass production, this enzyme activity belongs to advanced international standard, the better heat stability of enzyme.This enzyme Sargassum polysaccharides of can degrading effectively generates undersaturated alga oligosaccharide, for the high value trans-utilization of oceanic resources has been created condition.
Embodiment
Embodiment 1.
Bacterial strain N19-2 is kept and transfer on the solid slant culture base of test tube, this nutrient agar consists of: (grams per liter) starch 5, peptone 10, yeast powder 5, K
2HPO
41, MgSO
47H
2O 0.15, and NaCl 30, Na
2CO
310, agar powder 2%.Prepare with distilled water.
For each transfer, all agar slant was cultivated 2 days down at 35 ℃.Prepare microbial strain culture according to following steps then.
Embodiment 2
Transfer to the surface growth thing in the 250ml triangular flask that the 50ml seed culture medium is housed by agar surface, 35 ℃, shaking culture is 24 hours on 220 rev/mins of shaking tables, transfer then in 4 3000mL triangular flasks that the 1000ml seed culture medium is housed, cultivated under these conditions 24 hours, and be inoculated in 250 liters of fermentor tanks that 120 liters of fermention mediums are housed and ferment.Fermention medium consist of (grams per liter) sodium alginate 12, soyflour 4.5, monosodium glutamate 4.5, yeast powder 7, K
2HPO
41.5, MgSO
47H
2O 0.3, and NaCl 30, Na
2CO
310, soya-bean oil 4ml, 35 ℃ ± 1 ℃ of leavening temperature, air flow 1: 0.75,400 rev/mins of stirring velocitys.In the fermenting process, thalli growth is about 8 hours lag period, enter logarithmic phase after, the thalline ramp, the enzyme amount increases thereupon, (32-48 hour) enzyme activity is the highest during stationary phase.Ferment and to put jar in 40 hours, more than the fermentation broth enzyme vigor 100u/ml.
Fermented liquid filters with the sheet frame that adds the filter paper plate, filter pressure 0-2Kg/cm2, filtrate is concentrated by the Hollow Fiber Ultrafiltration post, (molecular weight cut-off of post is 6000) need not pressure substantially, concentrate about 10 times after, with 10mM Na
2CO
3-NaHCO
3Damping fluid (pH10) washing concentrating enzyme liquid, and washing ultrafiltration post.The total recovery 85% of final enzyme.
Embodiment 3.
Bacterial strain N19-2 is inoculated in the 250ml triangular flask that the 50ml seed culture medium is housed from the inclined-plane, and 35 ℃ of shaking tables are cultivated 24 hours as primary seed solution.Inoculum size with 3% is equipped with one of primary seed solution access in the 3000ml triangular flask of 1000ml seed culture medium, and 37 ℃ of shaking tables are cultivated 24 hours as secondary seed solution.The secondary seed solution access is equipped with in 50 liters of seed fermentation jars of 25 liters of seed culture mediums, 35 ℃ ± 1 ℃ of leavening temperature, air flow 1: 1.0,450 rev/mins of stirring velocitys were fermented after 24 hours, it is transferred be equipped with in 1 ton of jar of 600 liters of fermention mediums.The consisting of of fermention medium (grams per liter): sodium alginate 12, yeast powder 7, soyflour 4.5, monosodium glutamate 4.5, K
2HPO
41.5, MgSO
47H
2O 0.1, and NaCl 30, Na
2CO
312, soya-bean oil 4ml/ liter, 35 ℃ of leavening temperatures, air flow 1: 0.75,240 rev/mins of stirring velocitys were fermented 40 hours, stopped fermentation, more than the fermentation broth enzyme vigor 100u/ml.
Fermented liquid filters with the sheet frame that adds the filter paper plate, and adds suitable diatomite as flocculating aids.Filtrate is carried out ultrafiltration and concentration as described in example 1, concentrate about 10 times, and total recovery is more than 85%.
Claims (5)
1. a salt Zymomonas mobilis N19-2 (Halomonas sp.N19-2), its culture presevation number is CGMCC No.0464.
2. method of producing the Sargassum polysaccharides lyase by microbial fermentation, this method may further comprise the steps:
(1) with the salt Zymomonas mobilis N19-2 of claim 1 in seed culture medium, under the culture condition that is fit to seed growth, carry out seed culture;
(2) culture that (1) is gathered in the crops under the culture condition that is fit to thalli growth, carries out fermentation culture in containing the fermention medium of sodium alginate;
(3) Shou Huo fermented liquid concentrates by Plate Filtration and Hollow Fiber Ultrafiltration, makes the alkaline mycose lyase.
3. method according to claim 2, wherein seed culture medium is to contain starch 5 grams, peptone 10 grams, yeast powder 5 grams, K in every liter of nutrient solution
2HPO
41 gram, MgSO
4-7H
2O 0.15 gram, NaCl 30 grams, Na
2CO
310 grams, fermention medium are to contain sodium alginate 5-20 gram, peptone 4.5 grams, yeast powder 5-7 gram, K in every liter of nutrient solution
2HPO
41-3 gram, MgSO
47H
2O0.1-0.3 gram, NaCl 10-50 gram, Na
2CO
38-15 gram, soya-bean oil 2-5ml.
4. method according to claim 2, the culture condition of wherein said suitable seed growth are 35 ℃ of temperature, 220 rev/mins of shaking speed, shaking culture 24 hours; The fermentation culture conditions of described suitable thalli growth is temperature 32-37 ℃, air flow 1: 0.5-1: 1.0, and rotating speed 200-450 rev/min, fermentation time is 32-48 hour.
5. method according to claim 2, wherein the alkaline mycose lyase optimal reaction pH of Huo Deing is 9.5, optimal reactive temperature is 55 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001211064A CN1170925C (en) | 2000-07-26 | 2000-07-26 | Fermentation production process of alkaline mycose lyase and microbe for producing the lyase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001211064A CN1170925C (en) | 2000-07-26 | 2000-07-26 | Fermentation production process of alkaline mycose lyase and microbe for producing the lyase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1335393A CN1335393A (en) | 2002-02-13 |
| CN1170925C true CN1170925C (en) | 2004-10-13 |
Family
ID=4588591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001211064A Expired - Fee Related CN1170925C (en) | 2000-07-26 | 2000-07-26 | Fermentation production process of alkaline mycose lyase and microbe for producing the lyase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1170925C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911315A (en) * | 2014-01-03 | 2014-07-09 | 中国科学院天津工业生物技术研究所 | Strain for producing alginate lyase and use thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012157814A1 (en) * | 2011-05-17 | 2012-11-22 | 전남대학교 산학협력단 | Metagenome library having alginate lyase activity and novel enzyme alydw |
| CN103525717B (en) * | 2013-06-04 | 2014-12-31 | 昆明理工大学 | Anaerobic alginate decomposing bacterium and application thereof |
| CN108410849A (en) * | 2018-03-09 | 2018-08-17 | 集美大学 | A kind of Multifunction fishing polysaccharides lyase gene and its application |
-
2000
- 2000-07-26 CN CNB001211064A patent/CN1170925C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911315A (en) * | 2014-01-03 | 2014-07-09 | 中国科学院天津工业生物技术研究所 | Strain for producing alginate lyase and use thereof |
| CN103911315B (en) * | 2014-01-03 | 2016-03-02 | 中国科学院天津工业生物技术研究所 | Bacterial strain and the application thereof of algin catenase are produced in one strain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1335393A (en) | 2002-02-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100410364C (en) | Gamma-polyglutamic acid generating bacterium and process of preparing gamma-polyglutamic acid therewith | |
| CN101608166B (en) | Flavobacterium strain and application thereof in generating agarase | |
| US20240102058A1 (en) | Caproate-producing bacterium with multiple substrate utilization capabilities and its applications | |
| CN104911125A (en) | Chitosanase production strain and application thereof | |
| CN101451113A (en) | Vibrio natriegens and method for producing agarase by using the same | |
| WO2012016445A1 (en) | Bacillus subtilis strain and uses thereof | |
| CN111484954A (en) | Pseudomonas nigricans for producing alginate lyase | |
| CN100451106C (en) | Sphingomonaspaucimobilis of high-yield gellan gum and use therefor | |
| Yokoi et al. | Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29 | |
| CN108410783A (en) | A kind of method of high-density cultivation of Escherichia coli fermenting and producing Glucosamine | |
| CN107118980B (en) | Microbacterium keratanolyticum MCDA02 from sea, and its enzyme production method and product | |
| CN1170925C (en) | Fermentation production process of alkaline mycose lyase and microbe for producing the lyase | |
| CN103146595B (en) | Bacillus subtilis and method for fermentation production of D- ribose | |
| CN101392231A (en) | Recombinant bacillus coli and method for producing PHB by using biomass material and the same | |
| CN112592914A (en) | Special green alga polysaccharide lyase and production process thereof | |
| CN102127515A (en) | Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) | |
| CN102433274B (en) | Isoptericola halotolerans capable of highly producing alginate lyase and application method for isoptericola halotolerans | |
| CN106834390B (en) | Method for improving yield activity stability in glycoprotein flocculant production through bacterial fermentation | |
| CN101555505B (en) | Application and method of using pseudomonas mendocina NK-01 for production of brown alginate oligosaccharides | |
| CN110093297A (en) | Nitrate reduction bacterium MCDA3-3 and its method for producing chitin deacetylase | |
| CN1053697C (en) | Fermentation method for producing D-ribose novel strain, and method for prepn. of D-ribose using said strain | |
| CN113234633B (en) | Strain for producing chitinase and application of strain in preparation of chitosan oligosaccharide | |
| CN115058352B (en) | Bacillus subtilis and method for producing agricultural gamma-polyglutamic acid by using same | |
| CN117229979B (en) | Extended microbubble strain for producing algin lyase and application thereof | |
| CN108130347A (en) | A kind of method for producing chitin oligo saccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20041013 |