CN103320417B - Low cost preparation method of deep sea elastase - Google Patents
Low cost preparation method of deep sea elastase Download PDFInfo
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- CN103320417B CN103320417B CN201310228836.2A CN201310228836A CN103320417B CN 103320417 B CN103320417 B CN 103320417B CN 201310228836 A CN201310228836 A CN 201310228836A CN 103320417 B CN103320417 B CN 103320417B
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Abstract
The invention relates to a low cost preparation method of deep sea elastase, and the steps are as following: (1) inoculating deep sea bacteria into a liquid seed medium for culture to obtain a seed nutrient solution; inoculating the seed nutrient solution into a fermentation medium for fermentation culture, then taking the supernatant to make a broth; (3) performing overnight dialysis, centrifugation and DEAE-Sepharose Fast Flow chromatography and then gradient washing out to the broth, and taking the eluate of the first eluting peak to prepare the deep sea bacteria elastase. In the invention, elastin laminin is replaced by scalded sorta as a byproduct of a slaughterhouse and is used for enzyme production fermentation of bacterial strain CF6-2 extracellular elastase, therefore the fermentation cost is reduced by more than 70%, and the method has good environment benefit and social benefit.
Description
Technical field
The present invention relates to the low cost preparation method of a kind of deep-sea elastoser, particularly a kind of method of preparing deep-sea bacterium elastoser with Scalded Aorta fermentation, belongs to technical field of biotechnology.
Background technology
Elastoser (elastase) is a kind of to decompose the wide spectrum proteolytic ferment of insoluble elastin matter as feature, mainly be distributed in due to elastin in the reticular tissue such as blood vessel, lung and skin, therefore elastoser can be used as the biochemical drug of the aspects such as treatment hyperlipidaemia, control atherosclerosis, elimination skin eschar, chip, treatment is definite, safe and reliable; Also can be used for foodstuffs industry and cosmetic industry.And because the peptide section partial sequence of elastin and gram positive bacterium peptidoglycan has similarity, therefore elastoser sometimes also can be used as and has the N,O-Diacetylmuramidase that splits bacterium activity.N,O-Diacetylmuramidase can be used as natural food preservatives, now be widely used in anticorrosion in fishery products, meat product, cake, pure mellow wine, cooking wine and beverage, can also insert in milk powder, make cow's milk people emulsification, to suppress the existence of spoilage microorganisms in enteron aisle, promote directly or indirectly the propagation of bifidus bacillus in enteron aisle simultaneously.N,O-Diacetylmuramidase also has multiple pharmacological effect, and it has antibacterial, antiviral, antineoplastic effect, and current its indication of medical N,O-Diacetylmuramidase is hemorrhage, blood urine, bloody sputum and rhinitis etc.
On the earth, 80% biology is present in ocean, and therefore Living marine resources total amount is huge.Current research shows, marine microorganism is because of its unique living environment, and the proteolytic enzyme of secretion may have special character, is indicating and may have potential use.Elastoser research both at home and abroad for marine microorganism secretion is little, and overall development utilizes degree very low.Bacterium (Pseudoalteromonas sp.) CF6-2 separates and obtains from 2441 meters of dark marine bottom sediments of South China Sea.Research shows, this bacterial strain can be secreted taking elastin as inductor a kind of elastoser.The structure of proteolytic enzyme and the research of character to CF6-2 secretion show, novel proteolytic enzyme of Gai Meishi M23 family, elastin and peptidoglycan are all had to very high degrading activity, treatment bacterium infect and high blood pressure disease etc. medical aspect and the aspect such as makeup all there is good application potential.But it is higher to utilize elastin to carry out the fermentation costs of this enzyme, and this has had a strong impact on the Application and Development of this enzyme.Therefore, the low cost fermentation technique of the elastoser of invention Pseudoalteromonas sp.CF6-2 secretion will lay the foundation for the application and development of this enzyme.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of low cost preparation method of deep-sea bacterium elastoser is provided.
Technical solution of the present invention is as follows:
A low cost preparation method for deep-sea bacterium elastoser, step is as follows:
(1) by deep-sea bacterium (Pseudoalteromonas sp.) CF6-2 inoculation in liquid seed culture medium, shaking culture 12～15 hours under 15～18 DEG C of conditions, obtain seed culture fluid;
Described liquid seed culture medium component is as follows, is weight part:
0.8～1.0 part of peptone, 0.5～0.75 part of yeast powder, 100 parts of artificial seawaters, pH is 7.5～8.0;
(2) seed culture fluid by 1～5% volume percent, step (1) being made is inoculated in fermention medium, and after fermentation culture, centrifuging and taking supernatant, makes fermented liquid;
Described fermention medium component is as follows, is weight part:
0.8～1.2 part, core barrel powder, 0.1～0.5 part of yeast powder, CaCl
20.002～0.01 part, Na
2hPO
40.005～0.02 part, 100 parts of artificial seawaters, pH is 8.0;
Described core barrel powder is to be made after degreasing, freeze-drying, grinding, mistake 20 mesh sieves by Scalded Aorta;
(3) step (2) is made to fermented liquid through the 50mM of pH9.0 Tris-HCl damping fluid dialyzed overnight, centrifugal, supernatant liquor taking 2ml/min speed through carrying out gradient elution by concentration as the NaCl of 0～0.3M after DEAE-Sepharose Fast Flow chromatography column, get the elutriant of first elution peak, make deep-sea bacterium elastoser.
Preferred according to the present invention, deep-sea bacterium (Pseudoalteromonas sp.) the CF6-2 bacterial strain in described step (1) is to be under 15～20 DEG C of conditions in temperature, makes after 1～2 day in solid medium activation culture.
Further preferred according to the present invention, above-mentioned solid medium component is as follows, is weight part:
0.8～1.0 part of peptone, 0.5～0.75 part of yeast powder, 1.0～1.5 parts, agar, 100 parts of artificial seawaters, pH is 7.5～8.0;
Preferred according to the present invention, the fermentation culture in described step (2), temperature is 18～20 DEG C, the time is 26～30h.
Preferred according to the present invention, the centrifugal condition in described step (2) is: 5 DEG C of centrifugal 10min of 10000g.
Preferred according to the present invention, the centrifugal condition in described step (3) is: 5 DEG C of centrifugal 15min of 10000g.
Preferred according to the present invention, in described step (3), separation and purification chromatography column used is of a size of (55mm × 10cm), and cleansing temp is 4～6 DEG C.
Deep-sea bacterium described in above-mentioned steps (1) (Pseudoalteromonas sp.) CF6-2 strains separation is from South China Sea settling, be preserved in Chinese Typical Representative culture collection center on July 27th, 2010, address: Wuhan University's school of life and health sciences preservation center, its culture presevation is numbered CCTCC M2010189.
Degreasing in the preparation of core barrel powder, freeze-drying, grinding, mistake 20 mesh sieves all adopt this area conventional steps condition.
Beneficial effect
1, the present invention replaces elastin with slaughterhouse's byproduct Scalded Aorta, carries out the product enzymic fermentation of bacterial isolates CF6-2 Extracellular elastase, and fermentation costs reduces more than 70%;
2, the present invention adopts the fermentation condition of optimization, makes the work of bacterial strain CF6-2 extracellular enzyme reach 100.02 ± 9.0U/mL, and compared with prior art extracellular enzyme work has improved 1 times;
3, Scalded Aorta is slaughterhouse's byproduct, is difficult to digested and degraded, and the present invention carries out the fermentation of bacterium CF6-2 Extracellular elastase with Scalded Aorta, not only reduce fermentation costs, and has good environmental benefit and social benefit.
Brief description of the drawings
The graph of a relation that Fig. 1, core barrel powder consumption and deep-sea bacterium Extracellular elastase enzyme are lived;
The graph of a relation that Fig. 2, leavening temperature and deep-sea bacterium Extracellular elastase enzyme are lived;
The graph of a relation that Fig. 3, fermentation time and deep-sea bacterium Extracellular elastase enzyme are lived;
The growth curve of bacterium (A) and product enzyme curve (B) figure under Fig. 4, the most adaptable method.
Embodiment
Below in conjunction with Figure of description, technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
Core barrel powder, preparation method thereof is as follows:
The fat of fresh Scalded Aorta outer wall is removed clean, is cut into the bulk of about 1 square centimeter size, after dry 48 hours, use sander pulverize through vacuum freeze drier, obtain the core barrel powder used that ferments after 20 mesh sieves, in-20 DEG C of preservations.
Deep-sea bacterium (Pseudoalteromonas sp.) CF6-2 bacterial strain is purchased from Chinese Typical Representative culture collection center, and culture presevation is numbered CCTCC M2010189.
Embodiment 1
A low cost preparation method for deep-sea bacterium elastoser, step is as follows:
(1) deep-sea bacterium (Pseudoalteromonas sp.) CF6-2 bacterial strain, is under 20 DEG C of conditions in temperature, in solid medium activation culture 1 day, is then inoculated in liquid seed culture medium, and shaking culture 12 hours under 15 DEG C of conditions obtains seed culture fluid;
Described liquid seed culture medium component is as follows, is weight part:
0.8 part of peptone, 0.5 part of yeast powder, 100 parts of artificial seawaters, pH is 7.5;
(2) seed culture fluid by 1% volume percent, step (1) being made is inoculated in fermention medium, is 18 DEG C in temperature, and the time is, after 26h condition bottom fermentation is cultivated, to get supernatant at 5 DEG C of centrifugal 10min of 10000g, makes fermented liquid;
Described fermention medium component is as follows, is weight part:
0.8 part, core barrel powder, 0.1 part of yeast powder, CaCl
20.002 part, Na
2hPO
40.005 part, 100 parts of artificial seawaters, pH is 8.0;
(3) step (2) is made to fermented liquid dialyzed overnight to the 50mM Tris-HCl damping fluid of pH9.0, after in 5 DEG C of centrifugal 15min of 10000g, supernatant liquor with 2ml/min speed through carrying out gradient elution with 0M NaCl after DEAE-Sepharose Fast Flow chromatography column, chromatography column is of a size of 55mm × 10cm, cleansing temp is 4 DEG C, get the elutriant of first elution peak, make deep-sea bacterium elastoser.
Above-mentioned solid medium component is as follows, is weight part:
0.8 part of peptone, 0.5 part of yeast powder, 1.0 parts, agar, 100 parts of artificial seawaters, pH is 7.5.
Embodiment 2
A low cost preparation method for deep-sea bacterium elastoser, step is as follows:
(1) deep-sea bacterium (Pseudoalteromonas sp.) CF6-2 bacterial strain, is under 15 DEG C of conditions in temperature, in solid medium activation culture 2 days, shaking culture 15 hours under 18 DEG C of conditions, obtains seed culture fluid;
Described liquid seed culture medium component is as follows, is weight part:
1.0 parts of peptones, 0.75 part of yeast powder, 100 parts of artificial seawaters, pH is 8.0;
(2) seed culture fluid by 5% volume percent, step (1) being made is inoculated in fermention medium, is 20 DEG C in temperature, and the time is, after 30h condition bottom fermentation is cultivated, to get supernatant at 5 DEG C of centrifugal 10min of 10000g, makes fermented liquid;
Described fermention medium component is as follows, is weight part:
1.2 parts, core barrel powder, 0.5 part of yeast powder, CaCl
20.01 part, Na
2hPO
40.02 part, 100 parts of artificial seawaters, pH is 8.0;
(3) step (2) is made to fermented liquid dialyzed overnight to the 50mM Tris-HCl damping fluid of pH9.0, after in 5 DEG C of centrifugal 15min of 10000g, supernatant liquor with 2ml/min speed through carrying out gradient elution with 0.3M NaCl after DEAE-Sepharose Fast Flow chromatography column, chromatography column is of a size of 55mm × 10cm, cleansing temp is 6 DEG C, get the elutriant of first elution peak, make deep-sea bacterium elastoser.
Above-mentioned solid medium component is as follows, is weight part:
1.0 parts of peptones, 0.75 part of yeast powder, 1.5 parts, agar, 100 parts of artificial seawaters, pH is 8.0.
Test example 1
Different substrates reduce the mensuration of deep-sea bacterium elastoser fermentation costs effect:
The substrate of aseptic different content is joined in the substratum of bacterial strain CF6-2 to induction bacterium secretion Extracellular elastase.The induction effect of different substrates is as shown in table 1.
The impact of the different inductors of table 1. on bacterial strain CF6-2 fermentation product elastoser
As can be seen from Table 1, from the positive control of 0.3% elastin, can find out under elastin induction, reach higher level since the work of 36h enzyme, and rise slowly, the enzyme the highest time alive appears at 72h, is 46.9 ± 3.9U/mL, and the work of 84h enzyme starts to decrease.
There are inducing action in core barrel, lung, tendon and Chang Deng animal tissues to the secretion of deep-sea elastoser (pseudoalterin), but except core barrel can meet or exceed the induction level of elastin, the inducibility of remaining tissue is all lower, this may be because its hetero-organization Elastic protein content is lower, is not enough to induction and produces enough enzymes.Elastin can reach 40% in core barrel dry weight, therefore select 2.5% fresh core barrel can reach the induction effect of 0.3% elastin powder: in front 60h, the enzyme of core barrel induction is lived all a little less than elastin, but live and also reach maximum 54.6 ± 2.4U/mL at the enzyme of 72h core barrel induction, exceed the effect of elastin, this may be because the elastin in earlier fermentation core barrel due to by other tissue encapsulation wherein, be not easy to be utilized, later stage is along with the digestion of tissue, thereby elastin is released the efficient utilization that is conducive to bacterial strain, therefore enzyme is lived and is raise fast.During to 84h, the fermentation broth enzyme of core barrel induction is lived and is also started to decline.In other cheap protein substrates, except the inducing action of extractum carnis can exceed 10U/mL, remaining does not nearly all have inducing action, and what in extractum carnis, bring into play inducing action may be also the elastin in its composition.
At present, purify elastin market price 400 yuan/more than g, and core barrel is as the byproduct in slaughterhouse, conventionally only regarded food materials by barbecue industry, the market price of fresh Scalded Aorta is in about 40 yuan/kg, we use through degreasing, freeze-drying, grind and sieve after the cost of core barrel powder in 1 yuan/g left and right.Therefore pipe belt replaces elastin can significantly save the production cost of pseudoalterin diligently, is beneficial to its large-scale commercial production.
Test example 2
The impact that different fermentations condition is lived on a kind of deep-sea elastoser extracellular enzyme:
Control its dependent variable, only change respectively substratum pipe core consumption, culture temperature and incubation time and study the impact of different factors on pseudoalterin output, result is if Fig. 1 is to as shown in Fig. 4.
As can be seen from Figure 1 the yield effect of from 0.6% to 1.2% couple of pseudoalterin of core barrel powder is little, basic held stationary after yield of enzyme rising, and in the time that core barrel powder is increased to 1.0%, it is maximum that output reaches, and the output that exceedes enzyme after 1.2% obviously reduces; Different culture temperature also can affect the output of pseudoalterin, as can be seen from Figure 2 the suitableeest product enzyme temperature of bacterial strain CF6-2 between 17.5 to 22.5 ° of C, the secretion that is all unfavorable for enzyme too high or too low for temperature.Fig. 3 has reflected the variation of yield of enzyme under different incubation times.Increase to the straight line alive of enzyme between 25h at 15h, reach the highest when 25h, after 25h, enzyme is lived and is occurred small size decline, but maintains higher level always.
Fermentation process after optimizing is to adopt the core barrel powder of preparation as inductor, 0.8～1.2 part, core barrel powder, 0.1～0.5 part of yeast powder, CaCl
20.002～0.01 part, Na
2hPO
40.005～0.02 part, 100 parts of artificial seawaters, pH is 8.0; Result is as Fig. 4,18～20 DEG C of fermentations.Bacterial strain CF6-2 enters logarithmic phase after cultivation 5h, reaches stationary phase after 15h, and in fermented liquid, cell concentration reaches peak, substantially remains on this level within 20h afterwards, slightly declines to 35h.Fermentation broth enzyme work increases fast from 7.5h, reaches maximum 100.02 ± 9.0U/mL at 26.5h, is that 46.9 ± 3.9U/mL is doubled than the extracellular enzyme work in the situation of inducing with 0.3% elastin in the past.
Claims (2)
1. a preparation method for deep-sea bacterium elastoser, step is as follows:
(1) by deep-sea bacterium (
pseudoalteromonassp.) CF6-2 inoculation is in liquid seed culture medium, and shaking culture 12～15 hours under 15～18 DEG C of conditions, obtains seed culture fluid;
Described deep-sea bacterium (
pseudoalteromonassp.) CF6-2 strains separation, from South China Sea settling, is preserved in Chinese Typical Representative culture collection center, address on July 27th, 2010: Wuhan University's school of life and health sciences preservation center, and its culture presevation is numbered CCTCC M 2010189;
Described liquid seed culture medium component is as follows, is weight part:
0.8～1.0 part of peptone, 0.5～0.75 part of yeast powder, 100 parts of artificial seawaters, pH is 7.5～8.0;
(2) seed culture fluid by 1～5% volume percent, step (1) being made is inoculated in fermention medium, and after fermentation culture, centrifuging and taking supernatant, makes fermented liquid;
Described fermention medium component is as follows, is weight part:
0.8～1.2 part, core barrel powder, 0.1～0.5 part of yeast powder, CaCl
20.002～0.01 part, Na
2hPO
40.005～0.02 part, 100 parts of artificial seawaters, pH is 8.0;
Described core barrel powder is to be made after degreasing, freeze-drying, grinding, mistake 20 mesh sieves by Scalded Aorta;
(3) step (2) is made to the 50 mM Tris-HCl damping fluid dialyzed overnights of fermented liquid through pH 9.0, centrifugal, supernatant liquor taking 2 ml/min speed through carrying out gradient elution by concentration as the NaCl of 0～0.3 M after DEAE-Sepharose Fast Flow chromatography column, get the elutriant of first elution peak, make deep-sea bacterium elastoser.
2. preparation method as claimed in claim 1, is characterized in that, deep-sea bacterium in described step (1) (
pseudoalteromonassp.) CF6-2 bacterial strain is to be under 15～20 DEG C of conditions in temperature, makes after 1～2 day in solid medium activation culture.
3
.preparation method as claimed in claim 2, is characterized in that, above-mentioned solid medium component is as follows, is weight part:
0.8～1.0 part of peptone, 0.5～0.75 part of yeast powder, 1.0～1.5 parts, agar, 100 parts of artificial seawaters, pH is 7.5～8.0.
4
.preparation method as claimed in claim 1, is characterized in that, the fermentation culture in described step (2), and temperature is 18～20 DEG C, the time is 26～30 h.
5
.preparation method as claimed in claim 1, is characterized in that, the centrifugal condition in described step (2) is: 5 DEG C of 10000 centrifugal 10 min of g.
6
.preparation method as claimed in claim 1, is characterized in that, the centrifugal condition in described step (3) is: 5 DEG C of 10000 centrifugal 15 min of g.
7
.preparation method as claimed in claim 1, is characterized in that, in described step (3), separation and purification chromatography column used is of a size of 55 mm × 10 cm, and cleansing temp is 4～6 DEG C.
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