CN103255086B - The chitosan oligosaccharide that bacillus and its caused chitosan enzyme and the enzyme hydrolysis obtain - Google Patents
The chitosan oligosaccharide that bacillus and its caused chitosan enzyme and the enzyme hydrolysis obtain Download PDFInfo
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- CN103255086B CN103255086B CN201310147265.XA CN201310147265A CN103255086B CN 103255086 B CN103255086 B CN 103255086B CN 201310147265 A CN201310147265 A CN 201310147265A CN 103255086 B CN103255086 B CN 103255086B
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Abstract
The present invention relates to a kind of strain and application thereof, also relates to the chitosan enzyme that the strain is produced and the chitosan oligosaccharide obtained by the enzyme hydrolysis.Chitosan enzyme activity used in the present invention is high, Substratspezifitaet is strong, and can degrade obtained number-average molecular weight using the selectivity chitosan enzyme of the present invention as 1246.38, and the chitosan oligosaccharide of the degree of polymerization 46, gained chitosan oligosaccharide purity is high, has higher bioactivity.
Description
Technical field
The present invention relates to a kind of strain and application thereof, chitosan enzyme that the strain produced is also related to and by the enzyme hydrolysis
The chitosan oligosaccharide of acquisition.
Background technology
Chitosan is formed by N- acetyl group glucose polymerisations, is second largest large biological molecule polysaccharide in nature, nearly 20 years
To have caused extensive concern both domestic and external, substantial amounts of research is carried out.A variety of physiologically actives of chitosan have been found at present, and
Development and application are able in the numerous areas such as health food, biological medicine, daily-use chemical industry, environmental protection, but due to chitosan not
Water and other many inorganic solvents are dissolved in, limit it in multi-field development and application.Chitosan oligosaccharide is the catabolite of chitosan,
The oligosaccharide that the degree of polymerization being made up of the 2- Glucosamines of β-Isosorbide-5-Nitrae glucosides key connection is 2~20, has compared with chitosan
Relative molecular mass is small, good water solubility, bioactivity are high, the advantages that being easy to absorb, having a wide range of application.Chitosan oligosaccharide have antibacterial,
The functions such as anti-oxidant, antitumor, enhancing immunity of organism, it is a variety of to be widely used in medicine, food, beverage, cosmetics and chemical industry etc.
In field.In numerous degraded chitosans obtain the method for chitosan oligosaccharide, enzymatic isolation method is the degraded side being most widely used in recent years
Method.Most chitosan enzyme is with the chitosan of internal-cutting way hydrolyzable moiety acetylation.The height of chitosan oligosaccharide bioactivity with
Its degree of polymerization has close ties, and research shows, the degree of polymerization is 5~6 chitosan oligosaccharide bioactivity highest.At present in chitosan oligosaccharide system
In standby and application study, the chitosan oligosaccharide degree of polymerization is difficult to control, and is mostly the mixture of wide polymerization scope, and its bioactivity is unstable
It is fixed.The key and bottleneck that research about chitosan enzyme has been produced and applied as chitosan oligosaccharide with further utilization.Pass through
The superior strain for screening the selectivity chitosan enzyme obtained produces high-quality chitosan enzyme, and utilizes selectivity chitosan enzyme degraded shell
Glycan has the advantages that reaction is gentle, and easy to control, easily separated processing is free from environmental pollution, and its lytic activity is high.
The content of the invention
To solve the deficiencies in the prior art, the selectivity shell that the present invention provides one plant of fermentation and can obtain high activity gathers
Carbohydrase, bacillus of the degree of polymerization for the chitosan oligosaccharide of 4~6 high bioactivity can be obtained by the chitosan enzyme hydrolyzing chitosan
（Bacillus sp.）.
For achieving the above object, bacillus of the invention（Bacillus sp.）Dg belongs to bacillus, its in
On December 21st, 2012 is in the common micro-organisms center preservation of China Microbiological preservation administration committee, deposit number CGMCC
No.7024。
The bacterial strain size is 0.5-0.7 × 1.2-2.8um, terminal spore, no pod membrane, amphitrichous, Gram-positive bacillus.
It is in colony characteristicses on well-grown in PDA culture medium, PDA culture medium：Bacterium colony is thicker, surface is smooth, easily provoke, it is matt,
Light milky white is opaque, neat in edge.Optimum growth temperature is 36 ± 1 DEG C.Facultative anaerobic bacteria.Strain fermentation glucose, the fruit
Sugar, maltose production acid, it is impossible to produce acid using lactose, arabinose, xylose, agarose, galactolipin and sucrose；Indole test sun
Property, methyl red test is positive；V-P negatives；Hydrogen sulfide production test is positive；Sole carbon source can be used as by the use of citrate.
Simultaneously the invention provides the purposes of the bacterial strain, it is applied is producing chitosan enzyme by inducer fermentation of chitosan
In.
The invention further relates to a kind of chitosan enzyme, its bacillus by deposit number for CGMCC No.7024 simultaneously
（Bacillus sp.）Dg, fermented and produced as inducer using chitosan.
As the restriction to above-mentioned technical proposal, by bacillus（Bacillus sp.）Dg is using chitosan as inducer
Fermentation is produced in chitosan enzyme, and condition of enzyme production is that chitosan content is 0.5%-2%, and beef extract content is 0.25-0.35%, temperature
For 30-40 DEG C, inoculum concentration 5%-15%.
As the further restriction to such scheme, its optimal condition of enzyme production is that chitosan content is 1%, beef extract content
For 0.3%, temperature is 30 DEG C, inoculum concentration 15%.
As the restriction to chitosan enzyme reaction condition, the reaction temperature of the chitosan enzyme is 35-55 DEG C, pH 4.5-6.
As the restriction to such scheme, the optimal reactive temperature of the chitosan enzyme is 45 DEG C, pH 5.5.
In addition, present invention also offers a kind of chitosan oligosaccharide, the chitosan oligosaccharide is gathered by chitosan enzyme as described above degraded shell
Sugar obtains, and the chitosan oligosaccharide degree of polymerization is 4-6.
In above-mentioned technical proposal, the Selective agar medium using chitosan as sole carbon source is utilized, is sieved using hydrolysis method
Selecting the bacterial strain Dg obtained to have, enzymatic productivity is strong, producing chitosan enzyme activity, high and enzyme catabolite is living for degree of polymerization 4-6 height
Property chitosan oligosaccharide the features such as, ensure that by the present invention a large amount of selectivity chitosan enzymes can be prepared with relatively low cost, enter
And obtain high activity chitosan oligosaccharide.
During selectivity chitosan enzyme being obtained using the bacterial strain Dg fermentations that deposit number is GMCC No.7024, fermentation
Condition determine foundation be：Four principal elements for influenceing thalline producing enzyme vigor are followed successively by：Beef extract content, chitosan content,
Temperature, inoculum concentration.Beef extract content is to influence the primary factor of enzymatic activity, and fermentation process can be caused when beef extract content is too low
The deficiency of thalline nutrient matrix, so as to influence thalli growth and produce chitosan enzyme；Beef extract too high levels can cause culture medium
Carbon-nitrogen ratio is unbalance in formula, so as to cause zymotic fluid pH substantial deviation Liquid fermentation conditions, causes enzyme activity relatively low, while can also
The stability of chitosan enzyme in zymotic fluid is influenceed, and it is uneconomical；Chitosan be influence enzymatic activity the second key factor, chitosan
Enzyme is a kind of induced enzyme, needs to add a certain amount of chitosan in the fermentation medium and does inducer to improve yield of enzyme；Fermentation temperature
Degree influences thalli growth and producing enzyme, and the too low thalli growth of temperature and producing enzyme are slower, the easy aging of the too high thalline of temperature, and are unfavorable for producing
Enzyme；Inoculum concentration is too low, the Extending culture time, reduces the productivity ratio of fermentation tank；Inoculum concentration crosses conference and causes dissolved oxygen insufficient, influences to produce
Thing synthesizes；And metabolic waste can be excessively produced, it is also uneconomical.
When enzymatic chitosan hydrolyzate reaction temperature is too low, enzymatic reaction is slower, less efficient；When reaction temperature is too high,
The stability of chitosan enzyme decreases, at 70 DEG C, enzyme complete deactivation after 30min.Reaction system pH is too high too low to be influenceed
Enzyme activity, and then enzymatic reaction is influenceed, when pH value of reaction system is less than or equal to 4.0, during more than or equal to 7.0, the chitosan enzyme is lost
Enzyme activity.
In chitosan oligosaccharide preparation and application study, the chitosan oligosaccharide degree of polymerization is difficult to control, and is mostly the mixed of wide polymerization scope
Compound, its biologically active labile.Heretofore described selectivity chitosan enzyme enzymolysis shell is determined using acetylacetone method to gather
Chitosan oligosaccharide prepared by sugar, its number-average molecular weight are 1246.38, degree of polymerization 4-6, and its bioactivity is higher.
Brief description of the drawings
Below in conjunction with the accompanying drawings and embodiment is made further to describe in detail to the present invention：
Fig. 1 be enzyme liquid obtained by the embodiment of the present invention by SDS-PAGE methods, obtained chitosan enzyme gel electrophoresis figure；
Fig. 2 is chitosan enzyme Rate activity curve map under different temperatures；
Fig. 3 is chitosan enzyme Rate activity curve map under different pH.
Embodiment
Embodiment one
The present embodiment relate to the bacillus using deposit number as CGMCC No.7024（Bacillus sp.）Dg is sent out
The method that ferment prepares chitosan enzyme, this method comprise the following steps：
1st, actication of culture and fermentation
By the Dg inoculations of preservation on PDA slant mediums, cultivated 24 hours under the conditions of 37 DEG C, transfer two repeatedly
It is secondary, activated.Then, the strain activated is accessed in PDA liquid medium, 37 DEG C, prepared by 180rpm shaking table cultures 18h
Seed liquor.Finally, seed liquor is linked into fermentation medium by 15% inoculum concentration, 30 DEG C, 180rpm shaking table cultures 24h.
The formula of above-mentioned culture medium, conventional PDA culture medium can be used, in the present embodiment：
Inclined-plane seed culture medium（PDA culture medium）：Potato 200g（Peeling）, glucose 30g, peptone 2g, agar
20g, K2HP040.5g, MgS040.5g, VB1 10mg, distilled water 1000mL.
PDA liquid medium：With inclined-plane seed culture medium, but do not add agar.
In the fermentation medium (%, w/v) that the present embodiment uses, conventional inorganic salts and trace element can be used.It is worth
Illustrate, the content of beef extract and chitosan influences notable on producing enzyme in culture medium.In the present embodiment, fermentation medium
The formula of (%, w/v)：NaCl、KCl、MgS04·7H20 each 0.5%, K2HP040.075%, FeSO40.001%, peptone
0.85%, beef extract 0.3%, chitosan 1%, pH 7.5.
2nd, chitosan enzyme is extracted
By zymotic fluid through 5000rpm, 4 DEG C of low-temperature centrifugation 20min, crude enzyme liquid is obtained.Chitosan enzyme activity is in crude enzyme liquid
2000U/ml。
The enzyme activity determination of chitosan：The enzyme amount changed into 1 μ g chitosans of catalysis per minute required for chitosan oligosaccharide（Ml or
mg）As an enzyme-activity unit U.With every ml（Or mg）Enzyme activity unit number contained by enzyme liquid is specific activity of enzyme.
In the present embodiment, the measure of enzyme activity is with the following method：It is separately added into 50g's 3% in 100mL triangular flask
Chitosan gum liquid solution, is separately added into 5mL crude enzyme liquids after being preheated to 45 DEG C, 45 DEG C, reacted under the conditions of 180rpm, fixed
When sample, 1mol/L NaOH regulation pH to 8.0 is added dropwise, boiling water bath 20min, centrifuging and taking precipitation, 105 DEG C are dried to constant weight, claim
Sediment weight is measured, enzyme activity is finally calculated according to the decrement of precipitation.
Obtained crude enzyme liquid is precipitated with 60% alcohol chromatography, the crude product of chitosan enzyme is obtained after drying.
3rd, the determination of chitosan enzyme relative molecular weight
First, 500mL crude enzyme liquids are filtered with core filter, obtains removing the chitosan enzyme solution of thalline.
Then, the chitosan enzyme solution for removing thalline is carried out being classified alcohol analysis, it is chitosan enzyme to take 60% alcohol chromatography precipitation, is obtained
Dissolved again with distilled water after chitosan enzyme precipitation.
Using discontinuous SDS-PAGE electrophoresis, obtained chitosan enzyme gel electrophoresis figure, as shown in figure 1, in figure, 1 is pure
Enzyme liquid after change, 2 be standard protein sample, according to the relative migration distance reference standard protein of sample protein matter（Cow's serum
Albumen）Relative migration distance, the relative molecular mass for determining chitosan enzyme is 41.7kD.
4th, the determination of chitosan enzyme optimum reaction conditionses
（1）The measure of optimal reactive temperature
By the enzyme liquid of preparation and chitosan substrate（3% chitosan colloidal sol, its pH value are 5.0）Reaction, temperature are set respectively
For 25 DEG C, 35 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, remaining enzyme activity is determined, determines the optimal reactive temperature of the chitosan enzyme, it is determined
Curve is as shown in Figure 2.
As seen from Figure 2, being raised with temperature, the activity of chitosan enzyme is first raised and reduced afterwards, when temperature is 45 DEG C, enzyme
Work reaches highest.Therefore the optimal reactive temperature of the chitosan enzyme is 45 DEG C.
（2）The measure of optimal reaction pH value
By the chitosan substrate that the enzyme liquid of preparation and pH are 4.0,4.5,5.0,5.5,6.0,6.5 and 7.0（3% chitosan
Colloidal sol）In 45 DEG C of reactions, remaining enzyme activity is determined, detects the optimum pH of this chitosan enzyme reaction, it determines curve such as Fig. 3 institutes
Show, from the figure 3, it may be seen that with the rise of pH value, enzyme activity is first raised and reduced afterwards, when pH value is 5.5, enzyme activity highest, therefore it is most suitable anti-
It is 5.5 to answer pH value.
Embodiment two
The present embodiment is related to the preparation of chitosan oligosaccharide, is that the chitosan enzyme degraded chitosan obtained with embodiment one obtains, its
Preparation method is as follows：
3% chitosan gum liquid solution pH5.5 is preheated to 45 DEG C, adds 200 lists by every milliliter of 3% chitosan gum liquid solution afterwards
Position enzyme activity adds chitosan enzyme, 180rpm enzymolysis 2.5h, and 1mol/L NaOH are added dropwise and adjust pH to 8.0 terminating reactions, 4000 r/min
Centrifugation 20 minutes, supernatant is carried out to be classified alcohol analysis, takes 70% alcohol chromatography to precipitate, lyophilized precipitation is chitosan oligosaccharide.
To the chitosan oligosaccharide of acquisition produced above, molecular weight determination is carried out using acetylacetone method, its step is as follows：
The drafting of standard curve：Precision measures 30 μ g/ml GluNH2HCl solution 0,1.0,2.0,3.0,4.0,
5.Oml, it is respectively placed in tool plug test tube, respectively adds water to 5.Oml, then add acetylacetone,2,4-pentanedione solution 1mL respectively, shakes up, put boiling water bath
Middle heating 25min, take out, put in cold water and cool down, then add DMABA solution 1ml, add absolute ethyl alcohol to be shaken up, 60 DEG C of water-baths to lOmL
Middle insulation 1h, lets cool, and using 0 pipe as blank, A values is determined at 525nm wavelength, with GluNH2HCl micromoles number is ordinate,
A values are abscissa, draw standard curve.Obtaining calibration curve equation is：Y=0.8554x-0.0293, R2= 0.9992。
The ml of 100 μ g/ml chitosan oligosaccharides solution of accurate measuring 3 is placed in tool plug test tube, and remaining step is same as above, in wavelength 525nm
Place's measure A values.The molar concentration of chitosan oligosaccharide solution is calculated according to standard curve, the equal molecule of number of chitosan oligosaccharide is calculated according to formula
Amount.
Number-average molecular weight M=m/n of chitosan oligosaccharide
In formula：M is chitosan oligosaccharide quality, and n is the molal quantity of chitosan oligosaccharide.
It is 0.316 that chitosan oligosaccharide solution measures A values at 525nm, brings calibration curve equation into and obtains n=0.24 μm ol, then the shell
Oligosaccharides number-average molecular weight M=1246.38.The degree of polymerization for calculating the chitosan oligosaccharide accordingly is 4-6.
In summary, chitosan enzyme activity height, the Substratspezifitaet used in the present invention are strong, and utilize the selectivity of the present invention
Chitosan enzyme can degrade obtained number-average molecular weight as 1246.38, degree of polymerization 4-6 chitosan oligosaccharide, and gained chitosan oligosaccharide purity is high, tool
There is higher bioactivity.
Claims (3)
- A kind of 1. preparation method of chitosan enzyme, it is characterised in that：Described chitosan enzyme is by bacillus (Bacillus Sp.) fermented and produced as inducer using chitosan, the deposit number of wherein bacillus (Bacillus sp.) is：CGMCC No.7024。
- 2. the preparation method of chitosan enzyme according to claim 1, it is characterised in that：Condition of enzyme production is that chitosan content is 0.5%-2%, beef extract content are 0.25-0.35%, and temperature is 30-40 DEG C, inoculum concentration 5%-15%.
- 3. the preparation method of chitosan enzyme according to claim 1, it is characterised in that：Optimal condition of enzyme production contains for chitosan Measure as 1%, beef extract content is 0.3%, and temperature is 30 DEG C, inoculum concentration 15%.
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| CN104371989A (en) * | 2014-11-07 | 2015-02-25 | 中泰和（北京）科技发展有限公司 | Chitosanase and method for producing chitosan oligosaccharide by using same |
| CN106047964A (en) * | 2016-06-16 | 2016-10-26 | 领先生物农业股份有限公司 | Method for producing chitooligosaccharide from chitosan by enzymatic hydrolysis |
| CN106754829B (en) * | 2016-12-06 | 2020-10-16 | 鲁东大学 | Method for producing chitosanase by using bacillus HS17 fermentation and application thereof |
| CN108441440B (en) * | 2018-01-25 | 2021-03-23 | 山东省农业科学院农产品研究所 | Bacillus cereus 116 and application thereof |
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| CN102851239A (en) * | 2012-08-22 | 2013-01-02 | 黄河三角洲京博化工研究院有限公司 | Chitosanase producing strain and chitosan production method by using the same |
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| CN102851239A (en) * | 2012-08-22 | 2013-01-02 | 黄河三角洲京博化工研究院有限公司 | Chitosanase producing strain and chitosan production method by using the same |
Non-Patent Citations (2)
| Title |
|---|
| "聚合度4～6壳寡糖的制备及其活性研究";袁建平 等;《广东农业科学》;20121231(第8期);摘要 * |
| "芽孢杆菌Bacillus sp.S-1壳聚糖酶基因的克隆与序列分析";孙玉英 等;《中国生物工程杂志》;20091231;第29卷(第5期);摘要，第73页左栏最后一段 * |
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