CN102732462B - Renibacterium salmoninarum and enzyme produced thereby - Google Patents
Renibacterium salmoninarum and enzyme produced thereby Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a high-yield extracellular chitosan digestive enzyme marine bacteria Renibacterium Sp.QD1 and its separation and purification conditions and enzymatic properties of the high-yield extracellular chitosan digestive enzyme CSN-A. The invention is characterized in that a bacterial strain QD1 is subjected to fermentation culture, the activity of a fermented supernatant can reach 300-400 U/ml, a crude enzyme solution is subjected to ammonium sulfate precipitation and hydrophobic chromatography to obtain 28kDAchitosan digestive enzyme CSN-A. The most suitable temperature for degrading chitosan by enzyme is 50 DEG C, the optimal pH value is 5.6-5.8, and F<3+>, Al<3+>, Cu<2+> and SDS has strong inhibition effect on CSN-A enzyme activity, Mn<2+> can obviously activate CSN-A, a CSN-A degraded chitosan end product takes 2 glycosyl and 3 glycosyl as main components. The enzyme activity of the bacterial strain QD1 can reach 300-400U/ml broth under the condition suitable for industrial production. The bacterial strain of the invention solves the problems of low output and expensive price of the bacterial strain generated by the Chitosanase, and is capable of possessing great application prospect and greatly promoting the relative product utilization of chitosan and the like.
Description
Technical field
The invention belongs to microbe to screen and applied technical field.Be specifically related to the enzyme of a strain salmon Renibacterium and production thereof, namely a plant height produces the marine bacteria Renibacterium Salmoninarum QD1 of the outer chitoanase of born of the same parents and the degradation of chitosan enzyme CSN-A that produces thereof.
Background technology:
Chitin (chitin) claim again chitin, is that occurring in nature content is only second to cellulosic second largest class natural high moleculer eompound, approximately 1,000,000,000 tons of year biosynthesizing.It extensively is present in the cell walls of the arthropodan shells such as shrimp, crab and insects and part higher plant and fungi.Chitosan (chitosan) is the product that chitin is sloughed part or all of ethanoyl, is a class by 2-amino-2-DG and 2-acetylaminohydroxyphenylarsonic acid 2-DG (＜40%) monomer by straight-chain polysaccharide that β-the Isosorbide-5-Nitrae glycosidic link is formed by connecting.Compare with chitin, chitosan contains the more active primary amino of character, and it is uniquely in natural polysaccharide to contain cationic high molecular polymer, has different physiological roles, as germ resistance, one-tenth colloidality and film-forming properties etc., thereby has important using value in a plurality of fields.Chitoanase is β-Isosorbide-5-Nitrae-glycosidic link fracture of a class catalytic amino glucose, thus the glycoside hydrolase of specificity degrade chitosan.Chitoanase is of many uses, and on the one hand, chitoanase can be for the preparation of the oligochitosan with unique physiologically active; On the other hand, chitoanase itself can also as biological preservative, can improve the resistance against diseases of plant; At last, chitoanase occurs in the form of the self-dissolving of organism, cell walls, also has important purposes aspect the fundamental researchs such as Nutrition and Metabolism of soil microorganisms.
From 1973 found chitoanase first in the fungus and bacterium nutrient solution since, people had found the existence of chitoanase in the microorganism of the monoids such as many bacteriums, actinomycetes and fungi, but the activity of these chitoanases is mostly lower than 10U/ml.According to bibliographical information, Sun Yu English in 2007 etc. have reported that the chitoanase of a strain from ocean Microbacterium sp.OU01, its vigor reach 118U/ml, and the vigor of the aspergillus of the reports such as old little pretty young woman in 2004 after optimizing reaches 197U/ml.At present existing a plurality of chitoanases have been carried out commercialization, and its price is all more expensive, and the food grade chitoanase price of producing as Zhengzhou fine horse great waves chemical industry company limited is 460 yuan/liter.In sum, there is following shortcoming in the research of chitoanase at present, and the strain enzyme-producing ability is low, enzyme activity is not high, bacterial strain that be suitable for suitability for industrialized production is few, fermentation costs is high.Therefore, high vigor, chitoanase and microorganisms bacterial strain thereof have important market application foreground cheaply.
Summary of the invention:
The chitoanase that the purpose of this invention is to provide a strain salmon Renibacterium and production thereof, namely a plant height produces the bacterial strain of the outer chitoanase of born of the same parents, and the chitoanase of producing, thereby makes up existing technical deficiency.
Chitoanase producing bacterial strain provided by the invention is that the salmon Renibacterium belongs to marine bacteria Renibacterium Sp.QD1, the Chinese Typical Representative culture collection center that is positioned at Wuhan City, Hubei Province Wuchang District Wuhan University, preserving number: CCTCC NO:M 2011226 have been deposited on June 30th, 2011.
Bacterial strain of the present invention is used for producing shell 2 sugar (few 2 sugar of shell) and/or shell 3 sugar (few 3 sugar of shell).
Bacterial strain of the present invention can be secreted highly active degradation of chitosan enzyme CSN-A in a large number, is fit to suitability for industrialized production, and producing the suitableeest fermentation time of chitoanase is 40-42hr.
It is 25-28 ℃ that bacterial strain of the present invention produces the chitoanase optimum temperuture.
Above-mentioned chitoanase CSN-A from QD1 strain fermentation supernatant liquor separation and purification to, have following character:
Degradation of chitosan enzyme CSN-A, its SDS-PAGE electrophoresis molecular size range is 28kD.
Above-mentioned chitoanase, its effect substrate is chitosan.
Above-mentioned chitoanase, the optimum temperuture of degrade chitosan are 50 ℃.
Above-mentioned chitoanase, temperature-stable scope are 0-25 ℃.
Above-mentioned chitoanase, the optimal pH of degrade chitosan is 5.6.
Above-mentioned chitoanase, pH stable range are pH5-10.
Above-mentioned chitoanase, its inhibitor are Fe
3+, Al
3+, Cu
2+And SDS, promotor is Mn
2+
The end product of above-mentioned glycanase degrade chitosan is shell 2 sugar and shell 3 sugar.
Bacterium producing multi enzyme preparation involved in the present invention is through reaching 300-400U/ml to its vigor of its fermentation condition optimization, it is the higher bacterial strain of hitherto reported yield of enzyme, this bacterial strain has advantages of that also fermentation period is short, fermentation costs is low in addition, and chitoanase involved in the present invention has higher pH stability, indicated its potential value in industrial application.
Description of drawings:
Fig. 1: in the present invention, bacterial strain uses therefor produces the temperature curve collection of illustrative plates of the outer chitoanase of born of the same parents;
Fig. 2: in the present invention, bacterial strain uses therefor produces the time gradient curve of the outer chitoanase of born of the same parents;
Fig. 3: in the present invention, bacterial strain uses therefor produces the SDS-PAGE electrophorogram of the outer chitoanase CSN-A of born of the same parents;
Fig. 4: bacterial strain uses therefor of the present invention produces the outer chitoanase CSN-A optimum temperuture of born of the same parents and temperature stability collection of illustrative plates;
Fig. 5: in the present invention, bacterial strain uses therefor produces the outer chitoanase CSN-A optimal pH of born of the same parents and pH stability collection of illustrative plates;
Fig. 6: in various ion pair the present invention, bacterial strain uses therefor produces the effect of vigor collection of illustrative plates of chitoanase CSN-A;
Fig. 7: during this is clearly demarcated, bacterial strain uses therefor produces the end product tlc analysis of chitoanase CSN-A degrade chitosan.
Embodiment:
Below in conjunction with embodiment, the screening of invention bacterial strain, the character of enzyme are described in detail.
Separation and the evaluation of embodiment 1 bacterial strain
gather seawater from the Jiaozhou Bay, 4 ℃ of preservations, following method is processed: with seawater after the centrifugal 10min of 5000rpm/min, resuspended with a small amount of seawater, solution is carried out directly being coated on after gradient dilution contain on 0.5% chitosan solid medium flat board, cultivated 2-3 days in 28 ℃, selection is with the bacterium colony of obvious hydrolysis, the picking mono-clonal is inoculated in the test tube that contains the 3ml liquid nutrient medium, 28 ℃ of 200rpm/min cultivate 12hr, nutrient solution is contained in 250 milliliters of triangular flasks of 50ml liquid nutrient medium in the access of 1% ratio, cultivated 1-2 days in 28 ℃ of 200rpm/min.Nutrient solution carries out enzyme activity determination after centrifugal, the bacterial strain of screening high stability, high vigor.Strain bacterium that wherein vigor is the highest through morphological analysis and 16SrDNA sequencing, is shown that this bacterial strain belongs to the salmon Renibacterium and belongs to, and gramstaining is positive, called after Renibacterium Salmoninarum QD1.
The optimization of embodiment 2 strain fermentation conditions
1) the bacterial strain QD1 optimization of suitable leavening temperature
The inoculation of incubated overnight is in the 100ml fermented liquid, carries out fermentation culture respectively at 18,25,30,37 ℃, and timing sampling is measured chitoanase and lived, and as shown in Figure 1, the suitableeest leavening temperature of QD1 bacterial strain is defined as 25 ~ 28 ℃.
2) the bacterial strain QD1 optimization of suitable fermentation time
The inoculation of incubated overnight is in the 100ml fermented liquid, cultivates in 28 ℃, and timing sampling is measured the chitoanase vigor, and as shown in Figure 2, bacterial strain QD1 produces chitoanase and peaks at 40-42hr.
According to above-mentioned optimal conditions, bacterial strain QD1 produces the chitoanase vigor can bring up to the 300-400U/ml fermented liquid.
The liquid culture based formulas of using is (g/L): chitosan colloid 5g, sodium-chlor 1g, yeast powder 1g.The enzyme activity determination method is optical absorption method, 0.9ml 1% chitosan gum liquid solution adds 1ml 0.2M acetate buffer solution (PH5.8), bathes in 50 ℃ of temperature, adds 100ul fermentation supernatant crude enzyme liquid, in 50 ℃ of reaction 10min, react and add DNS reagent 1.5ml after complete, boil 10min in boiling water bath, be diluted with water to the 25ml volume, get 5ml in the centrifugal 5min of 10000rpm/min, supernatant liquor is measured light absorption value in 520nm, according to the glucosamine typical curve, tries to achieve the reducing sugar amount.An enzyme activity unit (U) is defined as 50 ℃ of lower per minute catalysis and generates the required enzyme amount of reducing sugar that is equivalent to 1 μ mol glucosamine.The preparation of chitosan gum liquid solution, take the 1g chitosan, add 10ml1M hydrochloric acid, glass stick is stirred to plastic, adds the 70ml deionized water, and 75 ℃ of water bath heat preservations are stirred to whole dissolvings, transfer PH to 6.0 with 1M sodium hydroxide, 75 ℃ are incubated to white precipitate and all dissolve, and add the water constant volume to 100ml, standby after 121 ℃ of sterilizations.
The separation and purification that embodiment 3 bacterial strains produce chitoanase CSN-A
The 1:CSN-A separation and purification of enzyme
the bacterial strain QD1 of incubated overnight is inoculated in the 100ml fermented liquid, in 28 ℃ of fermentation culture 36hr, collect fermented liquid with 12000g, 4 ℃ of centrifugal 20min, supernatant liquor spends the night with 60% ammonium sulfate precipitation, 4 ℃ of centrifugal 30min of 12000rpm/min, precipitation is dissolved with 20mM phosphoric acid buffer pH6.8, standby after 0.22 μ m membrane filtration, enzyme liquid loading hydrophobic chromatography post after filtration, carry out gradient elution with deionized water, collect each elution peak, live as following the tracks of with chitoanase, measure respectively each elution peak, the Peak Activity of gained is single band through the PAGE electrophoresis showed, called after CSN-A.Specific activity is brought up to 1574U/mg from 199U/mg albumen, has improved 7.89 times, and the rate of recovery is 67%, sees Table 1.
Purification procedures and the result of table 1 chitoanase CSN-A
The zymologic property of embodiment 4 chitoanase CSN-A
1) molecular weight of chitoanase CSN-A is determined
Because the relative mobility of protein is directly proportional to the logarithm of molecular weight, therefore can measure by SDS-PAGE the molecular weight of target protein.According to the relative mobility of SDS-PAGE Plays molecular weight protein and target protein, the molecular size range that calculates the CSN-A enzyme is 28kD, as shown in Figure 3, and this size and its in the same size in gel chromatography.
2) optimum temperuture of chitoanase CSN-A and thermal stability determination
CSN-A after purifying carries out enzyme assay at different temperature (20,30,40,50,60,70 ℃), result is as shown in Fig. 4 A.The optimum temperuture of CSN-A is 50 ℃, and when lower than 30 ℃ or during higher than 70 ℃, enzymic activity significantly reduces, and is only 15% left and right under optimum temperuture.
The temperature stability analysis of enzyme refers to enzyme measure remnant enzyme activity by standard method after the lower insulation of certain temperature (0,10,20,30,40,50) 1h, and result as shown in Figure 4 B.CSN-A is stability is arranged below 30 ℃ preferably, when temperature reaches 50 ℃, and pure enzyme whole inactivations almost in 1h.
3) optimal pH of chitoanase CSN-A and pH Stability Determination
The method of CSN-A after purifying under different pH carried out enzyme assay, and result is as shown in Fig. 5 A.The enzymic activity of CSN-A is more responsive to pH, and optimal pH is 5.6.Lower than 5 or higher than 7 the time, enzymic activity significantly descends, be lower than 50% of optimal pH as pH.
The mensuration of pH stability is with the enzyme liquid of equivalent and 4 ℃ of placement 24h of damping fluid of different pH, measures remnant enzyme activity by standard method during mensuration, and result is as shown in Fig. 5 B.Result shows, it is stable preferably that CSN-A all can keep between pH5-10.
4) impact of metal ion on the CSN-A enzymic activity
Investigate respectively the various ion (Li of 1mmol/L
+, Mn
2+, Zn
2+, Cu
2+, Ca
2+, Ba
2+, Mg
2+, Ni
2+, Al
3+, Fe
3+) on the impact that enzyme is lived, the enzyme work of control group is decided to be 100%.As shown in Figure 6, Cu
2+, Fe
3+And Al
3+Enzyme work to CSN-A has strong restraining effect, and surfactant SDS is lived to enzyme in addition also significant restraining effect, and Mn
2+Work has significant activation to enzyme.
5) the end product thin layer chromatography analysis of chitoanase CSN-A degrade chitosan
The preparation of substrate (1%): according to 1 gram chitosan powder: the ratio of 10ml 1M HCl adds hydrochloric acid, and 75 ℃ of heated and stirred are spent the night, with 1M NaOH readjustment PH to 6.0, to heat while stirring in the readjustment process, with the deionized water added body amass to concentration of substrate be 1%.
Hydrolysis: adjusting enzyme liquid concentration is 100U/ml, and according to the volume ratio of enzyme liquid: substrate=1:9, in 55 ℃ of Water Under solution 1h, the spray-dried rear sealing of product is preserved.
Product Identification: get appropriate product dry powder, be mixed with solution with deionized water, utilize thin-layer chromatography (TLC) to identify the composition of product, developping agent is: propyl carbinol: glacial acetic acid: water: ammoniacal liquor=2:1:1:0.3, dry up with blower after the exhibition layer, thin layer plate is immersed developer (2g pentanoic, 2mL aniline, the dense HCl of 1ml and 10mL85% phosphoric acid are dissolved in 200mL acetone altogether), dry with blower after taking out rapidly, then electricity consumption bake oven is baked silica-gel plate, till colour developing.
Result as shown in Figure 7, wherein M is shell 2 sugar and shell 3 saccharide, A is undegradable chitosan, the end product of the CSN-A of B explanation as a result degrade chitosan is sugared and shell 3 sugar of shell 2.
The bacterial strain that the present invention obtains and the chitoanase CSN-A that obtains from this bacterial strain have good prospects for commercial application, can be used for suitability for industrialized production shell 2 sugar and shell 3 sugar.
Claims (3)
1. a salmon Renibacterium belongs to marine bacteria salmon Renibacterium (Renibacterium Salmoninarum) QD1, and its deposit number is CCTCC NO:M 2011226.
2. the application of salmon Renibacterium claimed in claim 1 (Renibacterium Salmoninarum) QD1, is characterized in that, is to produce chitobiose and/or chitotriose.
3. the application of salmon Renibacterium claimed in claim 1 (Renibacterium Salmoninarum) QD1 in producing chitoanase.
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| CN110195047B (en) * | 2019-06-19 | 2021-08-20 | 枣庄全鼎生物科技股份有限公司 | Chitosanase CsnT and application thereof |
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