CN106929456A - A kind of temmoku acinetobacter calcoaceticus MCDA01 and its method for preparing chitin deacetylase - Google Patents
A kind of temmoku acinetobacter calcoaceticus MCDA01 and its method for preparing chitin deacetylase Download PDFInfo
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Abstract
The invention discloses a kind of temmoku acinetobacter calcoaceticus MCDA01 (Acinetobacter schindleri) from ocean, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.13538, the bacterial strain is Gram-negative brevibacterium, without gemma, bacteria colony white, it is opaque, the smooth moistening in surface, slightly projection, colony edge has spiculation, bacterial strain is 30 DEG C in optimum growth temperature, the most suitable growth pH is 8.0, the NaCl concentration of the most suitable growth is 4%, the most suitable fermentation pH of the chitin deacetylase that MCDA01 bacterial strains are produced is 8.0, optimum temperature is 30 DEG C；Chitin deacetylase prepared by the present invention has greater activity in low temperature, energy and expense can be saved in commercial Application, the shitosan prepared using the enzymatic has that deacetylation is high, biological enzyme environmental protection, the green production of shitosan is realized, with very big economic benefit.
Description
Technical field
It is a kind of temmoku acinetobacter calcoaceticus from ocean the present invention relates to a kind of microorganism, particularly a kind of temmoku is motionless
Bacillus MCDA01 and its method for preparing chitin deacetylase.
Background technology
Chitin is called chitin, chitin, is the material that content of cellulose is only second in natural organic-compound, extensively
It is present in the cell membrane of the ectoskeleton of invertebrate and epidermis and fungi and algae.Chitin is by N-Acetyl-D-glucosamine
The straight chain macromolecular material being formed by connecting by β-Isosorbide-5-Nitrae glycosidic bond, is not dissolved in water and organic solvent, it is not easy to widely opened
Hair is utilized.As chitin deacetylation just can obtain shitosan more than 55%, its dissolubility increases therewith, and with anticancer, drop
The bioactivity such as low cholesterol, hypotensive, can be widely applied to the industries such as medicine, food, chemical industry, cosmetics, have it is very high should
With value and DEVELOPMENT PROSPECT.
At present, chitin is converted into the processing method of shitosan two kinds of chemical heat alkaline process and biological enzyme, wherein, chemistry
Thermokalite method has that the reaction time is long, alkali charge big, the defect of high cost, there is the harm of pollution environment, and due to chemical heat
Alkali hair processes its deacetylation and is difficult to control and causes unstable product quality, is unsuitable for large-scale production.Additionally, biological enzyme
The main chitin that digested using chitin deacetylase obtains shitosan, but current microorganism prepares chitin deacetylase and deposits
Producing enzyme vigor is low, deacetylated effect difference the problems such as, be badly in need of expanding the screening for giving birth to microbial resources to producing chitin deacetylase,
Meet chitosan biological enzyme process industrialized production needs.
The content of the invention
In order to be directed to that above-mentioned chemical heat alkaline process is present as not environmentally, product stability is poor, whard to control and biology enzyme
Defect that enzymatic activity that solution is present is low, deacetylated effect is poor, cost is high etc., the invention provides a kind of environmental protection, improves and takes off
The method of the chitin deacetylase of acetyl degree, concrete scheme is as follows：
A kind of temmoku acinetobacter calcoaceticus MCDA01, the bacterial strain is temmoku acinetobacter calcoaceticus (Acinetobacter schindleri)
MCDA01, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC
NO.13538。
Further, the MCDA01 is Gram-negative brevibacterium, and bacteria colony white, opaque without gemma, surface is smooth
Moistening, slightly projection, colony edge have spiculation.
A kind of method that temmoku acinetobacter calcoaceticus MCDA01 prepares chitin deacetylase, it is characterised in that the temmoku is not
The method that lever bacterium MCDA01 prepares chitin deacetylase specifically includes following steps：
(1) 2216E fluid nutrient mediums, seed culture medium, fermentation medium are prepared,
(2) bacterial strain MCDA01 is seeded to inclined-plane seed culture in 2216E fluid nutrient mediums, and is screened with screening and culturing medium
Target strain,
(3) the inclined-plane seed obtained in above-mentioned steps (2) is seeded in seed culture medium carries out Shaking culture, has cultivated
It is centrifuged 10min with 1000r/min after, supernatant as seed liquor is taken,
(4) seed liquor obtained in above-mentioned steps (3) is seeded into fermentation medium with 1% inoculum concentration carries out shaking flask training
Support, prepare chitin deacetylase,
(5) sample is measured chitin deacetylase vigor in taking above-mentioned steps (4) at interval of 2h, is provided with boiling water
The enzyme liquid control group of bath inactivation 15min.
Further, 2216E fluid nutrient medium constituents are in the step (1)：Peptone 0.5%, dusty yeast
0.1%, FePO40.01%, agar 2%, Chen Haishui configurations；Seed culture medium constituent is：Dusty yeast 0.1%, peptone
0.5%, NaCl 1%, Chen Haishui are configured；Fermentation medium is grouped and is divided into：Peptone 0.5%, glucose 0.2%, NH4SO4
0.3%, KH2PO40.15%, MgSO40.05%, powder chitin 1%, Chen Haishui is prepared.
Further, 2216E fluid nutrient mediums pH value is 7.0, inclined-plane culture 48h, the inclined-plane of acquisition in the step (2)
Seed can make screening and culturing medium produce yellow circle.
Further, seed liquor shake flask culture conditions are liquid amount 20% in the step (3), and rotating speed is 180r/min,
5 DEG C -45 DEG C of temperature, pH value is 3.0-11.0, and NaCl concentration is 0%-10%, and the time is 0-24h.
Further, fermentation culture conditions are liquid amount 20%, fermentation time 2h-96h, temperature 10 in the step (4)
DEG C -30 DEG C, fermentation initial ph value is 4.0-10.0, derivant 1%-5%.
Further, in the step (2) screening and culturing medium for component that pH value is 7.0 be tobacco brown spot pathogen 0.2%,
K2HPO40.07%, KH2PO40.03%, MgSO40.05%, to nitro acetanilide 0.02%, Chen Haishui configurations.
Further, in the step (5) enzyme activity identification, in test tube add 30 DEG C of 0.05mol/L, pH of pre-incubations
It is worth paranitroacetanilide aqueous solution 1mL, the enzyme liquid 1mL for 7.0 phosphate buffer 3mL, 200mg/L, in 30 DEG C of water-baths
15min, boiling water bath terminates enzymatic reaction, and 3000r/min centrifugation 10min determine the absorbance of supernatant.
The present invention compared with prior art, has the following advantages：
Bacterial strain of the present invention is the temmoku acinetobacter calcoaceticus being separated in the ooze in Yellow Sea of China Haizhou Wan marine site
MCDA01, wide material sources can produce yellow on the screening and culturing medium containing tobacco brown spot pathogen and to nitro acetanilide
Circle, azymic glucose aerogenesis, in NaCl concentration for 0%-10% grows, belongs to salt tolerant marine microorganism, is sent out using it
Ferment prepares chitin deacetylase, and it has high activity when in low temperature, and catalysis activity is higher under normal temperature, prepared by MCDA01 fermentations
Chitin deacetylase, be capable of achieving large-scale industrial production, meet the demand of the shitosan in market pair, not only deacetylation
Height, pollution-free material discharge, reaches the purpose of green production, and protects environment, reduces production cost, improves economic effect
Benefit.
Brief description of the drawings
Fig. 1 is bacterial strain MCDA01 gram chromosomes (× 1000)；
Fig. 2 is the discoloration loop graph that bacterial strain MCDA01 is formed in primary dcreening operation flat board；
Fig. 3 is the influence figure that temperature grows to bacterial strain MCDA01；
Wherein, ● 5 DEG C, 0 15 DEG C, 30 DEG C, ▲ 45 DEG C of 25 DEG C of ▼, ■；
Fig. 4 is the influence figure that pH grows to bacterial strain MCDA01；
Fig. 5 is the influence figure that NaCl concentration grows to bacterial strain MCDA01；
Fig. 6 is influence figure of the fermentation time to bacterial strain MCDA01 enzymatic productions；
Fig. 7 is influence figure of the temperature to bacterial strain MCDA01 enzymatic productions；
Fig. 8 is influence figures of the initial pH of culture medium to bacterial strain MCDA01 enzymatic productions；
Fig. 9 is influence figure of the sample-loading amount to bacterial strain MCDA01 enzymatic productions；
Figure 10 is influence of the powder chitin addition to bacterial strain MCDA01 enzymatic productions；
Figure 11 is influence of the temperature to bacterial strain MCDA01 chitin deacetylases activity；
Figure 12 is influence of the temperature to bacterial strain MCDA01 chitin deacetylase stability；
Wherein, ● 4 DEG C, 0 15 DEG C, ▲ 25 DEG C, 30 DEG C of ▼；
Figure 13 is influences of the pH to bacterial strain MCDA01 chitin deacetylases activity,
Wherein, ▲ pH5.0-pH8.0, ● pH8.0-pH9.0, zero pH 9.0-pH12.0；
Figure 14 is influences of the pH to bacterial strain MCDA01 chitin deacetylase stability,
Wherein, ▲ pH5.0-pH8.0, ● pH8.0-pH9.0, zero pH 9.0-pH12.0.
Biological material specimens preservation：
Temmoku acinetobacter calcoaceticus MCDA01 (Acinetobacter schindleri) is preserved in in January, 2017 No. 6
China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological
Culture Collection Center (CGMCC)), deposit number is CGMCC NO.13538, depositary institution address：Beijing
The institute 3 of city Chaoyang District North Star West Road 1.
Specific embodiment
Further to disclose technical scheme, the implementation method that the invention will now be described in detail with reference to the accompanying drawings：
Wherein, culture medium used in the present invention is：2216E fluid nutrient mediums：Peptone 0.5%, dusty yeast 0.1%,
FePO40.01%, agar 2%, Chen Haishui configurations, pH 7.0；Screening and culturing medium：Tobacco brown spot pathogen 0.2%, K2HPO4
0.07%, KH2PO40.03%, MgSO40.05%, to nitro acetanilide 0.02%, Chen Haishui configurations, pH 7.0；Kind
Sub- culture medium：Dusty yeast 0.1%, peptone 0.5%, NaCl 1%, Chen Haishui configuration, pH 7.0；Fermentation medium：Peptone
0.5%, glucose 0.2%, NH4SO40.3%, KH2PO40.15%, MgSO40.05%, powder chitin 1%, Chen Haishui
Prepare, pH 8.0.
Embodiment 1：
(1) characteristic of MCDA01
In form, the bacterial strain is Gram-negative brevibacterium, without gemma, as shown in Figure 1.Cultivated in LB solid cultures
After 48h, the white opaque shape of bacterium colony, the smooth moistening in its surface, slightly projection, colony edge has spiculation, is difficult to choose
Take.Yellow circle can be produced on the screening and culturing medium containing tobacco brown spot pathogen and to nitro acetanilide, referring to Fig. 2.Institute
Stating MCDA01 has physio-biochemical characteristics, as shown in table 1：
The physiological and biochemical test result of the MCDA01 bacterial strains of table 1
Note：+：It is positive；-：It is negative；
(2) culture of MCDA01：
The 2216E inclined-planes seed of bacterial strain MCDA01 is inoculated into seed culture medium carries out Shaking culture, wherein control temperature
30 DEG C of degree, rotating speed 180r/min, liquid amount 20% cultivates 24h.
In order to further probe into the influence that temperature grows to bacterial strain MCDA01, seed liquor is connected to 1% inoculum concentration
In 2216E fluid nutrient mediums, under the conditions of being 20% in rotating speed 180r/min, liquid amount, 24h is cultivated at different temperatures respectively,
Then its OD is determined600Value.From the figure 3, it may be seen that the bacterial strain is significantly reduced less than 5 DEG C and higher than 45 DEG C of speeds of growth, most suitable life
Temperature long is 30 DEG C.
In order to further probe into influence of the pH value to strain growth, in the scopes of pH 3.0-pH 11.0, temperature control 30
DEG C, in remaining condition, Shaking culture is carried out to MCDA01, bacterium well-grown in the range of pH 6.0-pH 9.0 is found, it is most suitable
Growth pH is 8.0, referring to Fig. 4.
In order to further probe into influence of the NaCl concentration to strain growth, the present invention configures 2216E culture mediums with distilled water,
Control NaCl scopes for 0%-10%, be then inoculated with the bacterium and cultivated under the conditions of optimum temperature and pH, find the bacterium in 0%-
Can be grown in the range of 10% NaCl concentration, the NaCl concentration of the most suitable growth is 4.0%, has certain growth without NaCl, is belonged to
Salt tolerant marine microorganism, referring to Fig. 5.
(3) fermentation MCDA01 prepares chitin deacetylase：
Seed liquor is seeded to fermentation medium, wherein 30 DEG C of temperature, liquid amount 20%, shaking flask rotating speed with 1% inoculum concentration
For 180r/min carries out fermentation 96h, enzyme activity is surveyed every 2h samplings, as a result show 72h producing enzyme highests, referring to Fig. 6.
In order to further probe into influence of the fermentation temperature to bacterial strain MCDA01 producing enzymes, the present invention is by seed liquor with 1% inoculation
Amount is seeded to fermentation medium, and wherein liquid amount is that 20%, Shaking culture rotating speed is 180r/min, is trained at different temperatures
72h is supported, production of enzyme reaches highest when as a result showing 25 DEG C, there is yield of enzyme higher at 15 DEG C, reaches the 80% of highest producing enzyme, joins
See Fig. 7.
In order to further probe into influences of the culture medium starting pH to bacterial strain MCDA01 producing enzymes, the present invention is by seed liquor with 1%
Inoculum concentration is seeded to fermentation medium, and it is 180r/ for 25 DEG C of temperature, liquid amount 20%, shake flask fermentation rotating speed to control fermentation condition
Min, the different initial pH of regulation fermentation medium, enzyme activity is determined after culture 72h, is as a result shown, with the rising enzyme of initial pH
Yield gradually increases, and when pH reaches 8.0, yield of enzyme reaches maximum, and production of enzyme is notable during less than pH 5.0 or higher than pH 10.0
Reduce, referring to Fig. 8.
In order to further probe into influence of the liquid amount to bacterial strain MCDA01 producing enzymes, the present invention is by seed liquor with 1% inoculum concentration
It is seeded to different liquid amount fermentation mediums, wherein fermentation condition is that 25 DEG C of temperature, rotating speed are 180r/min, is surveyed after culture 72h
Determine enzyme activity, liquid amount producing enzyme highest at 20% in 250mL triangular flasks is as a result shown, referring to Fig. 9.
In order to further determine that influence of the derivant to bacterial strain MCDA01 producing enzymes, the present invention is by seed liquor with 1% inoculum concentration
The fermentation medium of different chitin contents is seeded to, fermentation condition is controlled for 25 DEG C of temperature, liquid amount 20%, rotating speed are
180r/min, determines enzyme activity after culture 72h, yield of enzyme reaches maximum when chitin addition is 1%-5%, less than 1%
Or during higher than 5%, producing enzyme is significantly reduced, it is contemplated that economic cause, selection 1%, referring to Figure 10.
(4) chitin deacetylase activity determination method：
Added in test tube 30 DEG C of the 0.05mol/L pH of pre-incubation 7.0 phosphate buffer 3mL, 200mg/L to nitro second
Anilide aqueous solution 1mL, enzyme liquid 1mL, in 30 DEG C of water-bath 15min, boiling water bath terminates enzymatic reaction, 3000r/min centrifugations
10min, determines the absorbance of supernatant.It is control with the enzyme liquid for adding the same concentration boiling water bath inactivation 15min of 1mL.Wherein, enzyme
Unit (U) definition living：Enzyme amount required for producing 1 μ g paranitroanilinum per hour under the above-described reaction conditions is defined as an enzyme
Unit of activity.
First, influence of the temperature to bacterial strain MCDA01 chitin deacetylases activity and stability：In pH7.0,
In purifying chitin deacetylase vigor is determined under different temperatures in 0.05mmol/L phosphate buffers, determine enzyme is best suitable for work
With temperature, Figure 11 is as a result seen, the operative temperature that is best suitable for of enzyme is 30 DEG C, more than 50% relative enzyme is still kept at 15 DEG C and 45 DEG C
It is living.
By chitin deacetylase in pH7.0,0.05mol/L phosphate buffers respectively at being incubated respectively under different temperatures
0h-2.5h, then determines residual enzyme activity, determines enzyme temperature stability, as a result sees Figure 12, and enzyme is most stable at 4 DEG C, 25 DEG C
Half-life period is 1.5h.
Secondly, influences of the pH to bacterial strain MCDA01 chitin deacetylases activity and stability：Prepare different pH's
0.05mol/l buffer solutions, phosphate buffer (pH 5.0-pH 8.0), Tris-HCl buffer solutions (pH 8.0-pH 9.0), Gly-
NaOH buffer solutions (pH 9.0-pH 12.0).30 DEG C under the buffer solution of different pH with paranitroacetanilide be substrate determine
The vigor of enzyme, is as a result shown in Figure 13, and the optimal pH of enzyme is 8.0, has more than 80% enzymatic activity in pH 7.0-pH 10.0.
Enzyme is incubated 1h in the buffer solution of different pH respectively at 30 DEG C, then at 30 DEG C in the phosphate buffers of pH 7.0
Determine residual enzyme activity.Result is shown in Figure 14, and enzyme has more than 80% loudness enzyme activity in pH 7.0-pH 10.0, in pH 10.0
More than 40% enzymatic activity is kept, in acid condition, enzymatic activity is significantly reduced.
The above, specific embodiment only of the invention, but protection scope of the present invention is not limited thereto, and it is any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all contain
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (9)
1. a kind of temmoku acinetobacter calcoaceticus MCDA01, it is characterised in that the bacterial strain is temmoku acinetobacter calcoaceticus (Acinetobacter
Schindleri) MCDA01, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC NO.13538。
2. a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 1, it is characterised in that：The MCDA01 is gram
Negative brevibacterium, bacteria colony white, opaque without gemma, the smooth moistening in surface, slightly projection, colony edge have spiculation.
3. a kind of method that temmoku acinetobacter calcoaceticus MCDA01 prepares chitin deacetylase, it is characterised in that：The temmoku is motionless
The method that bacillus MCDA01 prepares chitin deacetylase specifically includes following steps：
(1) 2216E fluid nutrient mediums, seed culture medium, fermentation medium are prepared,
(2) bacterial strain MCDA01 is seeded to inclined-plane seed culture in 2216E fluid nutrient mediums, and target is screened with screening and culturing medium
Strain,
(3) the inclined-plane seed obtained in above-mentioned steps (2) is seeded in seed culture medium carries out Shaking culture, after the completion of culture
It is centrifuged 10min with 1000r/min, supernatant as seed liquor is taken,
(4) seed liquor obtained in above-mentioned steps (3) is seeded into fermentation medium with 1% inoculum concentration carries out Shaking culture, makes
Standby chitin deacetylase,
(5) sample is measured chitin deacetylase vigor in taking above-mentioned steps (4) at interval of 2h, is provided with boiling water bath and goes out
The enzyme liquid control group of 15min living.
4. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：In the step (1)
2216E fluid nutrient medium constituents are：Peptone 0.5%, dusty yeast 0.1%, FePO40.01%, agar 2% is old
Seawater is configured；
Seed culture medium constituent is：Dusty yeast 0.1%, peptone 0.5%, NaCl 1%, Chen Haishui configuration；
Fermentation medium is grouped and is divided into：Peptone 0.5%, glucose 0.2%, NH4SO40.3%, KH2PO40.15%,
MgSO40.05%, powder chitin 1%, Chen Haishui is prepared.
5. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：2216E fluid nutrient mediums pH value is 7.0 in the step (2), and inclined-plane culture 48h, the inclined-plane seed of acquisition can make
Screening and culturing medium produces yellow circle.
6. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：Seed liquor shake flask culture conditions are liquid amount 20% in the step (3), and rotating speed is 180r/min, 5 DEG C -45 of temperature
DEG C, pH value is 3.0-11.0, and NaCl concentration is 0%-10%, and the time is 0-24h.
7. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：Fermentation culture conditions are liquid amount 20%, fermentation time 2h-96h, 10 DEG C -30 DEG C of temperature, hair in the step (4)
Ferment initial ph value is 4.0-10.0, derivant 1%-5%.
8. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：In the step (2) screening and culturing medium for component that pH value is 7.0 be tobacco brown spot pathogen 0.2%, K2HPO4
0.07%, KH2PO40.03%, MgSO40.05%, to nitro acetanilide 0.02%, Chen Haishui configurations.
9. the method that a kind of temmoku acinetobacter calcoaceticus MCDA01 according to claim 3 prepares chitin deacetylase, it is special
Levy and be：The identification of enzyme activity in the step (5), it is 7.0 phosphorus that 30 DEG C of 0.05mol/L of pre-incubation, pH value are added in test tube
Paranitroacetanilide aqueous solution 1mL, the enzyme liquid 1mL of acid buffer 3mL, 200mg/L, in 30 DEG C of water-bath 15min, boiling water
Bath terminates enzymatic reaction, and 3000r/min centrifugation 10min determine the absorbance of supernatant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458022A (en) * | 2020-12-09 | 2021-03-09 | 吉林中粮生化有限公司 | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof |
| CN112458022B (en) * | 2020-12-09 | 2022-08-16 | 吉林中粮生化有限公司 | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof |
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| CN106929456B (en) | 2019-08-27 |
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