CN100419072C - Keratinase-proudicng bacterium and its preparation method - Google Patents
Keratinase-proudicng bacterium and its preparation method Download PDFInfo
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- CN100419072C CN100419072C CNB2005100487738A CN200510048773A CN100419072C CN 100419072 C CN100419072 C CN 100419072C CN B2005100487738 A CNB2005100487738 A CN B2005100487738A CN 200510048773 A CN200510048773 A CN 200510048773A CN 100419072 C CN100419072 C CN 100419072C
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Abstract
The present invention relates to a bacterium for producing keratinase and a preparation method thereof. The bacterium for producing keratinase is classified and named Bacillus subtilis, and the storage registration number of the bacterium is CGMCC No. 1505. A good bacterial strain for producing keratinase is sieved in the preparation method; the enzyme reaction of the coarse enzyme liquid of keratinase produced by the bacterial strain has an optimal pH value of about 7.5 to 8.0 and an optimal temperature of 50 to 60 DEG C, and the stability and the heat stability of the enzyme reaction are good; over 80% of the enzyme activity of the coarse enzyme liquid can be maintained in the pH range of 6.4 to 10 after heat is insulated for 1 hour under different pH conditions; about 90% of the residual enzyme activity of the coarse enzyme liquid can be maintained after heat is insulated for 30 minutes in a water bath at the temperature of 70DEG C; over 70% of the enzyme activity of the coarse enzymes liquid can be maintained after heat is insulated under for 120 minutes. Compared with reported keratinase, the keratinase of the present invention has a better heat resisting property and pH stability.
Description
Technical field
The present invention relates to generation bacterium of a kind of M-Zyme and preparation method thereof.
Background technology
Keratin sulfate (Keratin) is present in occurring in nature with the hairs of various animals, feather, hoof tips etc. in a large number as principal mode, is the very strong rigid albumen of a kind of resistance.Keratin molecule forms highly cross-linked three-dimensional structure by disulfide linkage, hydrogen bond, sat linkage and other cross linkage effect of Gelucystine, highly stable, water insoluble, salt, diluted acid and diluted alkaline also are difficult for being degraded by most protein lytic enzyme (as trypsinase, casease, stomach en-etc.).M-Zyme (Keratinase) is the enzyme of the degraded natural keratin that a class can be single-minded, can significantly improve keratic digestibility, and some M-Zyme also has hydrolytic action to the multiple proteins except that Keratin sulfate simultaneously.
M-Zyme can decompose keratic characteristic specifically, and it is had broad application prospects at aspects such as fodder industry, foodstuffs industry, leather processing, detergent industry, medicine industry and environmental improvements.In fodder industry, M-Zyme can be converted into feather, pig hair the like waste the feedstuff protein of high nutrition.Keratin sulfate is the main ingredient in the waste-feather that produces in the bird processing, its crude protein content is up to more than 80%, contain various total amino acid contents more than 70%, and be rich in the needed indispensable amino acid of multiple animal, it is a kind of feedstuff protein source that application prospect is arranged very much, but it forms so that the Gelucystine disulfide linkage is highly interconnected, can not be degraded by general proteolytic enzyme, therefore is difficult to be absorbed by animal.Problems such as though high temperature steaming or chemical treatment can improve proteinic digestibility, the existence power consumption is big, and the nutritional quality of product is easily destroyed, and the three wastes are difficult to be handled, and environmental pollution is serious.Utilize M-Zyme to address this problem effectively, its advantage is more economical, improves discarded keratic utilization ratio, and the product nutritive value is good simultaneously, and pollution-free.The feather resource of China is extremely abundant, the annual production few hundred thousand tonnes of, but this part good protein source fails to be utilized effectively at present, both caused the waste of resource, contaminate environment again, and China's feed protein resource is seriously deficient, according to China's feed industrial development planning, will reach 8,000,000 tons to the breach of feedstuff protein in 2010.Therefore, the development and utilization of feather protein feed resource has and important meaning.In addition, M-Zyme also has multiple industrial use, as in foodstuffs industry, can be used to produce high-nutrition foods such as fish sauce, meat peptone, or as the tenderization agent; In leather processing industries, its alternative common proteolytic enzyme or be used with common proteolytic enzyme improves treatment effect and reduces cost; Pharmaceutically, M-Zyme helps wound healing, removes scab and epithelium regeneration, is specifics of surgical operation and skin burn, wound or the like.
The research work of M-Zyme has the history of decades, and the many microorganisms of occurring in nature all can secrete M-Zyme, can decompose Keratin sulfate specifically, make it be degraded to polypeptide and amino acid.Since Ward in 1899 has reported that fungi horse onyx group capsule bacterium (Onygena equina) can be decomposed Keratin sulfate, kind of a microorganism has the keratic ability of degraded surplus successively finding 30, mainly comprise: dermatophytosis in the fungi and Candida albicans (Candida albicans), Bacillus licheniformis (Bacillus licheniformis) in the streptomycete in the actinomycetes (Streptomyces) and high temperature zygosaccharomyces (Thermonspora) and the bacterium and subtilis (Baccillus subtilis) etc.The bacterial classification that is used for fermentative production at present is mainly streptomyces fradiae (Streptomyces fradiae) and Bacillus licheniformis (Bacillus licheniformis).Nineteen ninety, the separation such as Williams of U.S. North carolina state university obtain producing high yield bacterium Bacillus licheniformis (Bacillus licheniformis) PWD-1 (ATCC 53757) of M-Zyme, extract the outer M-Zyme of born of the same parents that this bacterial classification of purifying produces, and its physico-chemical property carried out detailed research, this enzyme molecular weight is about 33kD, enzyme is all very stable in pH 5～12 scopes, the reaction optimum temperuture is 50 ℃, specific activity is 5990U/mg, the feather that high temperature steaming is not crossed but this M-Zyme can not be degraded, poor heat resistance, and can self-catalytic pyrolysis (Applied Environ.Microbio.58:3271-3275,1992).Agricultural University Of Jiangxi has carried out a series of research to streptomyces fradiae S-221, and they use the C that designs and improve voluntarily
2Liquid fermentation medium, the last enzyme work of fermented liquid reaches 66.2ug/ml, and this enzyme useful effect pH is 7-10, handles 0.5 hour down at 70 ℃, and its enzymic activity only maintains about 70% (Agricultural University Of Jiangxi's journal, 19 (4): 60-65,1997).The important meaning of keratic microbiological deterioration and utilization has more and more caused people's extensive interest.More external scholars' research work, M-Zyme higher structure, activity expression molecular basis and the research biologically of enzyme engineering equimolecular have been related to, mainly concentrate on the M-Zyme gene of Bacillus licheniformis, measured its encoding sequence and successfully clone and express.And the research of state's interior opposite angle proteolytic enzyme also rests on separation, screening, the separation and purification of M-Zyme and the research of physico-chemical property of producing the M-Zyme bacterium and the preliminary study of the mechanism of action of M-Zyme substantially.And the object of research is also extremely limited, mainly concentrates on the research to streptomyces fradiae.At home, produce M-Zyme for fermentation using bacteria and still belong to blank.
Summary of the invention
The purpose of this invention is to provide generation bacterium of a kind of M-Zyme and preparation method thereof, the M-Zyme enzymic activity that produces with this bacterium is good, has good stability and thermostability.
The microbe species that the present invention utilizes Yunnan to distribute reaches the resourceful advantage of keratin protein more, screen the strain excellent that produces M-Zyme, the optimal pH of its crude enzyme liquid enzyme reaction is about 8.0, optimum temperuture is 60 ℃, have good stability and thermostability, behind insulation 1h under the different pH conditions, in the scope of pH6.4-10, enzymic activity all maintains more than 80%.Behind the insulation 30min, residual enzyme is lived and is also had about 90% in 70 ℃ of water-baths; Behind the insulation 120min, enzymic activity still maintains more than 70%, and through preliminary evaluation classification called after subtilis (Bacillus subtilis), its preservation registration number is CGMCC No.1505.
The preparation method of this bacterial strain is:
1) separation of bacterial strain and screening: from the rubbish soil that is rich in feather keratin, water drain mud, sewage and poultry moving point soil sampling, carry out continuous enrichment culture as only nitrogen source with feather keratin.Soil sampling inserts enrichment culture liquid and adds the complete feather of 3-5 root, and in 37 ℃, 200r/min enrichment culture 3-10 days treats that feather is decomposed, and in its nutrient solution switching enrichment culture liquid, carries out the enrichment culture second time under similarity condition.Choose feather and decompose nutrient solution preferably, flat board coating separation dilution is carried out in the dilution back on the primary dcreening operation flat board routinely, places 37 ℃ of constant incubators to cultivate 1-2 days, watches the dull and stereotyped periphery of bacterial colonies that goes up whether to form the obvious transparent circle.Select bacterium colony line separation and purification 2-3 time of big and obvious transparent circle; Behind the purifying its bacterial strain is inserted the liquid fermentation medium shake flask fermentation, further primary dcreening operation and multiple sieve, final separation screening to decompose utilize the feather keratin ability strong, produce M-Zyme enzyme high bacterial strain alive;
2) this inoculation (the liquid fermentation medium of the feather of inoculating strain is not in contrast) in the liquid fermentation medium that contains feather, they are placed 37 ℃ simultaneously, 200r/min condition bottom fermentation is after 4 days, the plumage sprig that inoculates the liquid fermentation medium mesoptile of this bacterial strain all comes off, only surplus plumage stalk, and contrast liquid fermentation medium mesoptile is undecomposed.
The used substratum of separation screening is:
Enrichment medium (g/L): NH
4Cl 0.5, and NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O0.1, yeast extract paste 0.1, feather meal 10 (adds a small amount of sterilized complete feather, pH7.5) during inoculation.
Screening culture medium A (g/L): NH
4Cl 0.5, and NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O 0.1, yeast extract 0.1, casein 15, agar 18-20 (pH7.5).
Screening culture medium B (g/L): NaCl 10, and peptone 10, yeast extract 5, Azokeratin10, agar 18-20.
Liquid fermentation medium (g/L): NH
4Cl 0.5, and NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O 0.1, yeast extract 0.1, and feather meal 10 (adds a small amount of sterilized complete feather, pH7.5) during inoculation.
The preliminary evaluation of bacterial strain:
It is carried out morphological specificity, cultural characteristic, the preliminary evaluation of aspects such as Physiology and biochemistry mensuration according to " common bacteria system identification handbook " and " microbiology laboratory manual ".
Bacterial strain is the Gram-positive genus bacillus, has pod membrane and middle life or inferior terminal spore (see figure 2).It can be grown in 20～60 ℃ of temperature ranges, and optimum growth temperature is 37～50 ℃, and growth pH is 5～11, and the suitableeest growth pH is 7～8.The energy hydrolyzed starch, the NaCl growth of ability 7%, nitrate reduction, catalase experiment, V.P experiment, Citrate trianion and propionic salt utilization, nitrite reduction, gelatine liquefication, casein results of hydrolysis are positive, and methyl red experiment, hydrogen sulfide experiment, indoles experimental result are negative.This bacterial strain is bacillus (Bacillus sp) through preliminary evaluation.
The fermentation of bacterial strain:
The seed culture fluid access is equipped with in the 250ml triangular flask of 50ml feather fermention medium, places the speed governing of rotary type constant temperature to shake a bottle cabinet and carry out aerobic fermentation, rotating speed is 200r/min, and 37 ℃ or 50 ℃ fermented 3～4 days.Fermented liquid is centrifugal, gets supernatant liquor, places 4 ℃ of preservations.Used substratum is:
Seed culture medium (g/L): sodium-chlor 10, peptone 10, yeast extract paste 5, agar 20
Fermention medium (g/L): NH
4Cl 0.5, and NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O0.1, yeast extract 0.1, feather meal 10 (adds a small amount of sterilized complete feather, pH7.5) during inoculation.
Enzyme activity determination method and thick enzyme zymologic property:
1, the enzyme activity determination method (is seen: Lin, x, LeeC.-G., casale, E.S., andshih, J.C.H.1992.Purification and characterization of keratinase froma feather-degrading Bacillus licheniformisstrain..Appl.Environ.Microbin.58:3271-3275).
(1) determination step is as follows:
1. 20mg azo Keratin sulfate (Azokeratin) is dissolved in 3.2mL phosphoric acid buffer (pH8.0), and the concussion mixing is incubated 2min in advance in 50 ℃ of water-baths;
2. the enzyme liquid 0.8mL that adds suitable extension rate accurately is incubated 15min in 50 ℃ of water-baths;
3. add 10% trichoroacetic acid(TCA) 0.8mL, enzyme reaction is stopped;
It is 4. centrifugal that (10000r/min 6min) and under 450nm surveys light absorption value;
Contrast liquid adds 10% trichoroacetic acid(TCA) 0.8ml earlier, enzyme-added again liquid 0.8mL when 5. reacting;
Enzyme work is defined as: the reaction of every ml enzyme liquid after 15 minutes OD value to increase by 0.01 be 1U.
(2) azo Keratin sulfate pretreatment process: (see: Tomarelli, R.M., Charney J., andHarding M.L., The use of azoalbumin as a substrate in the colormetricdetermination of peptic and tryptic activity.J.Lab, Clin, Med., 34,428-433)
Solution A: the 50g Keratin sulfate be dissolved in 1L distilled water again with 10% sodium hydrogen carbonate solution 100mL mixing;
Solution B: the 0.025mol sulfanilic acid adds the 5mL5.0N sodium hydroxide solution after being dissolved in 200mL distilled water, adds 0.025mol Sodium Nitrite mixing then; Add 0.05molHCl (10mL 5.0N hydrochloric acid soln) and stir 2min; Add 0.05molNaOH (10mL5.0N sodium hydroxide solution) again and stir 5sec; With the solution A mixing,, place baking oven oven dry (not being higher than 60 ℃) to get final product again at last with after the mixed solution dialysed overnight.
2, thick enzyme zymologic property
(1) optimum temperuture of enzyme reaction and optimum pH
Make the azo Keratin sulfate in the damping fluid of pH8.0, (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃) carry out enzymatic reaction under differing temps, survey its enzyme and live gained result such as Fig. 3.The result shows that its enzyme reaction is that 50-70 ℃ of scope endoenzyme activity change is little in temperature, and optimum temperuture is 60 ℃.Prepare the phosphoric acid buffer of different pH values (4.4,5.6,6.4,7.2,7.6,8.0,8.6,9.0,10,11), substrate azo Keratin sulfate carries out enzymatic reaction under 60 ℃ in different pH damping fluids, surveys its enzyme and lives gained result such as Fig. 4.The result shows that at the pH of M-Zyme be in the 6.4-10 scope, and enzymic activity changes little, and optimal pH is 8.0.
(2) the pH stability test of M-Zyme
After the enzyme liquid of the damping fluid of different pH values (4.4,5.6,6.4,7.2,7.6,8.0,8.6,9.0,10,11) preparation placed 60 ℃ of water-baths insulation 60min respectively, survey its enzyme and live gained result such as Fig. 5.The result shows that this M-Zyme has better pH stability, handle in the pH6.4-8.0 scope that enzymic activity all maintains behind the 1h more than 80%.
(3) heat stability test of M-Zyme
Place 70 ℃ water-bath to be incubated 10min, 30min, 50min, 70min, 90min, 120min respectively, gained result such as Fig. 6 enzyme liquid.The result shows that this enzyme has better heat-resisting, handles 30min down at 70 ℃, and its residual enzyme is lived and also had about 90%; After handling 120min, enzymic activity still maintains more than 70%.
The present invention screens the strain excellent that produces M-Zyme, the optimal pH that this bacterial strain produces the enzyme reaction of M-Zyme crude enzyme liquid is about 7.5-8.0, optimum temperuture is 50-60 ℃, have good stability and thermostability, behind insulation 1h under the different pH conditions, in the scope of pH6.4-10, enzymic activity all maintains more than 80%.Behind the insulation 30min, residual enzyme is lived and is also had about 90% in 70 ℃ of water-baths; Behind the insulation 120min, enzymic activity still maintains more than 70%.Comparatively speaking, than the M-Zyme of having reported better heat-resistant quality and pH stability are arranged.
Description of drawings
Figure 1A and Figure 1B are the decomposition run figure of bacterial strain of the present invention to feather.
Fig. 2 is bacterial strain spore staining figure of the present invention.
Fig. 3 is the optimum temperuture graphic representation of enzyme reaction of the present invention.
Fig. 4 is the optimal pH graphic representation of enzyme reaction of the present invention.
Fig. 5 is the influence curve figure of pH of the present invention to enzyme stability.
Fig. 6 is the thermostability graphic representation of enzyme of the present invention.
The microbial preservation date of the present invention is on October 23rd, 2005, and depositary institution is called for short CGMCC, deposit number: CGMCC No.1505.
Embodiment
Embodiment:
The separation of bacterial strain and screening:
1) near slaughterhouse, ground, plants such as Yiliang, Yunnan, Jian Shui, Yuxi, is rich in the rubbish soil, water drain mud, sewage of feather keratin and poultry moving point soil sampling, carries out continuous enrichment culture as only nitrogen source with feather keratin.Soil sampling 5g inserts 50mL enrichment culture liquid and adds the sterilized complete feather of 3-5 root, and enrichment medium is by (g/L): NH
4Cl0.5, NaCl0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O 0.1, yeast extract paste 0.1, and feather meal 10 is formed, and in 37 ℃, 200r/min enrichment culture 3-10 days treats that feather is decomposed, and in its nutrient solution switching 50mL enrichment culture liquid, carries out the enrichment culture second time under similarity condition.
2) select for use above-mentioned feather to decompose nutrient solution preferably, dilute gradient (10 routinely
-1, 10
-2, 10
-3, 10
-4, 10
-5) dilution back carries out flat board coating and separate dilution on the screening flat board, place 37 ℃ of constant incubators to cultivate 1-2 days, watches dull and stereotyped upward periphery of bacterial colonies whether to form the obvious transparent circle.Select bacterium colony line separation and purification 2-3 time of big and obvious transparent circle, final separation screening utilizes to decomposing that the feather keratin ability is strong, product M-Zyme enzyme high bacterial strain alive.Here used screening culture medium is: screening culture medium A (g/L): NH
4Cl 0.5, and NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O 0.1, yeast extract 0.1, casein 15, agar 18-20 (pH7.5); Screening culture medium B (g/L): NaCl 10, and peptone 10, yeast extract 5, and Azokeratin 10, and agar 18-20 uses screening culture medium A earlier, uses screening culture medium B again, screens twice.
With this inoculation (the liquid fermentation medium of the feather of inoculating strain is not in contrast) in the liquid fermentation medium that contains feather, they are placed 37 ℃ simultaneously, 200r/min condition bottom fermentation is after 4 days, the plumage sprig that inoculates the liquid fermentation medium mesoptile of this bacterial strain all comes off, only surplus plumage stalk, and contrast liquid fermentation medium mesoptile is undecomposed, the results are shown in accompanying drawing 1.Figure 1A mesoptile is undecomposed; And the plumage sprig of Figure 1B mesoptile all comes off, only surplus plumage stalk.Liquid fermentation medium (g/L): NH
4Cl0.5, NaCl 0.5, K
2HPO
40.3, KH
2PO
40.4, MgCl
26H
2O 0.1, yeast extract 0.1, and feather meal 10 (adds the sterilized complete feather of a small amount of 3-5 root, pH7.5) during inoculation.
Claims (1)
1. the generation bacterium of a M-Zyme, its called after subtilis (Bacillussubtilis) of classifying, its preservation registration number is CGMCC No.1505.
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| CN102517235B (en) * | 2011-12-27 | 2013-01-16 | 湖南农业大学 | Bacillus subtilis |
| CN102703368A (en) * | 2012-04-10 | 2012-10-03 | 江南大学 | Genetically engineered strain capable of producing keratinase and application thereof |
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| CN103642739B (en) * | 2013-12-19 | 2015-09-30 | 江南大学 | Streptomyces aureus and the application method thereof of M-Zyme are produced in one strain |
| CN103898081B (en) * | 2014-04-22 | 2016-04-13 | 青岛蔚蓝生物集团有限公司 | A kind of M-Zyme mutant and application thereof |
| CN105018382B (en) * | 2015-07-27 | 2018-04-03 | 湖南省微生物研究院 | The Lu Te of one plant of production keratinase is situated between this streptomycete LT 2 and its application process |
| CN105199983B (en) * | 2015-09-23 | 2018-10-09 | 四川大学 | The application of one bacillus amyloliquefaciens and its protease of generation in terms of process hides depilation |
| CN105112344A (en) * | 2015-10-08 | 2015-12-02 | 江南大学 | Brevibacillus parabrevis producing keratinase and application thereof |
| CN105820979B (en) * | 2016-04-25 | 2019-04-19 | 湖北工业大学 | One plant of feather degradation bacteria strains and application |
| CN107118984B (en) * | 2017-05-09 | 2018-06-08 | 山东省农业科学院生物技术研究中心 | A kind of ferment tank technique of keratin degrading bacteria and its application in live pig loses hair or feathers |
| CN107384799B (en) * | 2017-07-24 | 2021-02-09 | 大连理工大学 | Screening method of strain for degrading feather and application method of strain in feather degradation |
| CN110747188B (en) * | 2019-11-18 | 2021-01-29 | 山东隆科特酶制剂有限公司 | Method for producing keratinase |
| CN110747128B (en) * | 2019-11-18 | 2021-01-22 | 山东隆科特酶制剂有限公司 | Bacillus licheniformis strain capable of producing keratinase in high yield and application thereof |
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