CN103642739B - Streptomyces aureus and the application method thereof of M-Zyme are produced in one strain - Google Patents
Streptomyces aureus and the application method thereof of M-Zyme are produced in one strain Download PDFInfo
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- CN103642739B CN103642739B CN201310701537.6A CN201310701537A CN103642739B CN 103642739 B CN103642739 B CN 103642739B CN 201310701537 A CN201310701537 A CN 201310701537A CN 103642739 B CN103642739 B CN 103642739B
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Abstract
The present invention discloses a strain Keratin sulfate producing bacterial strain and culture condition thereof and thick enzymatic property, belongs to microbial technology field.Through qualification, this bacterial strain is streptomyces aureus (Streptomyces aureofaciens), called after K13, is now preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.8047.Through initial optimization, the substratum of this bacterial strain is: wool 0.5-1%, glucose 4-8%, peptone 3-5%, SODIUMNITRATE 0.3%, dipotassium hydrogen phosphate 0.04%, sodium-chlor 0.05%, shaking table culture condition is: initial pH value is 10.0, inoculum size 10%, culture temperature 30 DEG C, rotating speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h, produce M-Zyme vigor and reach 44.2U/ml.This M-Zyme is middle temperature basic keratins enzyme, and optimum temperature is 50-60 DEG C, and optimal pH is 8.5, and this enzyme enzyme activity in alkalescence (pH7.0-10.0) environment is stablized.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a strain and produce the streptomyces aureus of M-Zyme and thick enzymatic property thereof.
Background technology
China produces a large amount of poultry feathers and useless time wool every year, and its main component is Keratin sulfate.Because Keratin sulfate exists a large amount of disulfide linkage, hydrogen bond and hydrophobic interaction, be not easy by general proteins enzymic hydrolysis, thus major part is not utilized, and what have even causes environmental pollution.M-Zyme can change poultry feather, useless wool degraded into soluble protein, polypeptide or amino acid, as medical material, animal-feed etc., turns waste into wealth, reduces environmental pollution.Many microorganisms can produce M-Zyme, as streptomycete, Zymomonas mobilis, genus bacillus etc.
A strain is the object of the present invention is to provide to produce the streptomyces aureus of M-Zyme, and its thick enzymatic property of preliminary study.
Summary of the invention
the method such as screening, qualification, training systern of M-Zyme producing bacterial strain is as follows:
(1) dull and stereotyped primary dcreening operation: rural area sheepfold gathers pedotheque near Southern Yangtze University, gets 1g soil sample and adds in 10ml physiological saline, prepare soil supension, dilution spread, in primary dcreening operation substratum plate, cultivates 3d for 30 DEG C.Larger single bacterium colony that picking can grow on separating plate, line is separated to single bacterium colony.
(2) shaking flask is sieved again: the bacterial strain that primary dcreening operation obtains, through shake flask fermentation, measures its supernatant liquor M-Zyme vigor, finds that No. 13 bacterium enzyme activities are higher.
(3) bacterial strain 16S rDNA identifies: extract STb gene, increase with bacterial 16 S rDNA universal primer, the gene order obtained is 1500 bp, through order-checking comparison, for streptomyces aureus (Streptomyces aureofaciens), called after K13, be deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on August 19th, 2013, preserving number is CGMCC No.8047.
(4) strain fermentation condition initial optimization: utilize single factor test and orthogonal experiment to be optimized the fermention medium of bacterial strain and fermentation condition, obtaining optimum medium formula is wool 0.5-1%, glucose 4-8%, peptone 3-5%, SODIUMNITRATE 0.3%, dipotassium hydrogen phosphate 0.04%, sodium-chlor 0.05%, shaking table culture condition is: initial pH value is 10.0, inoculum size 10%, culture temperature 30 DEG C, shaking speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h, produce M-Zyme vigor and reach 44.2U/ml.
(5) thick Characterizaton: have studied operative temperature and action pH respectively to the impact of this enzyme activity, find that the optimum temperature of this M-Zyme is 50-60 DEG C, optimal pH is 8.5, and this enzyme enzyme activity stable (± 10%) in alkalescence (pH7.0-10.0) environment.This enzyme is middle temperature basic keratins enzyme.
(6) measuring method of M-Zyme vigor: get a 18ml test tube, add pH Tris-HCl damping fluid 2.0ml, the wool powder substrate 10mg of crude enzyme liquid (fermented supernatant fluid) 1.0ml, 0.05mol/l successively, 37 DEG C of constant temperature oscillation water-bath reaction 1h, add the trichoroacetic acid(TCA) 2.0mL termination reaction of 0.4mol/l, centrifuging and taking supernatant liquor measures its light absorption value in 280nm.Compare with the reaction tubes adding trichoroacetic acid(TCA) before 37 DEG C of constant temperature oscillations.
Enzyme activity defines: in above-mentioned reaction system, it is 1 enzyme activity unit (U/ml) that the absorbance under 280nm increases by 0.01 unit.
Wherein the primary dcreening operation substratum described in step (1) is (g/L): wool powder 5, K
2hPO
41, NaCl 0.5, agar powder 20; Multiple sieve Medium of shaking flask fermentation described in step (2) is (g/L): wool powder 10, K
2hPO
40.4, NaCl 0.5.
Of the present invention
pseudomonas xanthomarinadN1 is denitrifying bacterium, can be widely used in the purification of various eutrophication water.
Embodiment
the selection systems of embodiment 1 M-Zyme producing bacterial strain
1, culture medium prescription
(1) primary dcreening operation substratum (g/l): wool powder 5, K
2hPO
41, NaCl 0.5, agar powder 20.
(2) seed liquid nutrient medium
Gao Shi substratum (g/l): Zulkovsky starch 20, KNO
31, NaCl 0.5, K
2hPO
40.5, M gSO
4 .7H
2o 0.5,
FeSO
4·7 H
2O 0.01。
LB substratum (g/l): peptone 10, yeast powder 5, sodium-chlor 10.
Czapek's solution (g/l): sucrose 30, NaNO
33, K
2hPO
41, M gSO
4 .7H
2o 0.5, KCl 0.5, FeSO
4 .7 H
2o
0.01。
(3) fermention medium (g/l): wool powder 10, K
2hPO
40.4, NaCl 0.5
2, dull and stereotyped primary dcreening operation and Keratin sulfate vitality test
Near Southern Yangtze University, rural area sheepfold gathers pedotheque, getting 1 g soil sample adds in 50 ml triangular flasks of 10 ml physiological saline and several granulated glass spherees, and shaking table vibrates 15 min after soil sample dispersion, leaves standstill 5 min, draw l ml soil supension to 9 ml diluent (stroke-physiological saline solution), obtain 10
-2extent of dilution suspension, is diluted to l0 by l0 times of dilution method successively
-7, obtained each extent of dilution soil supension thus.(placement is spent the night to primary dcreening operation culture medium flat plate to draw each extent of dilution suspension 0.1 ml respectively, without varied bacteria growing), in 30 DEG C of constant temperature culture 3d, with the transfering loop bacterium colony come in every shape that picking growth is larger one by one to new primary dcreening operation culture medium flat plate, line is separated to single bacterium colony repeatedly.
Through initial gross separation purifying, obtain 32 strain bacterium, wherein 13 strains are actinomycetes, and 18 strains are bacterium, and 1 strain is fungi, and the bacterial strain that growth selection is better continues to cultivate, and finally obtains 18 strain bacterium.
3, shaking flask is sieved again
The 18 strain bacterial strains that primary dcreening operation is obtained, Gao Shi substratum, LB substratum and czapek's solution is adopted to carry out cultivation activation preparation seed liquor respectively, fermention medium is seeded to by the inoculum size of 2%, 30 DEG C, 220 r/min cultivate 64 h, measure the vigor of fermented supernatant fluid M-Zyme, finding that No. 13 bacterium enzyme activities are higher, is 3.9 U/ml.
4,16S rDNA identifies
Extract STb gene, with the amplification of bacterial 16 S rDNA universal primer, the gene order obtained is 1500 bp, through order-checking comparison, with the 16S rDNA sequence homology of streptomyces aureus up to 99%, tentatively judge its as streptomyces aureus (
streptomyces aureofaciens), called after K13.
Embodiment 2 strain fermentation condition initial optimization
Utilize single factor test and orthogonal experiment to be optimized the fermention medium of bacterial strain and fermentation condition, obtaining optimum medium formula is wool 0.5-1%, glucose 4-8%, peptone 3-5%, SODIUMNITRATE 0.3%, dipotassium hydrogen phosphate 0.04%, sodium-chlor 0.05%, shaking table culture condition is: initial pH value is 10.0, inoculum size 10%, culture temperature 30 DEG C, shaking speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h.It produces M-Zyme vigor and reaches 44.2U/ml, improves 10.3 times before comparatively optimizing.
the thick Characterizaton of embodiment 3
Preliminary study produce the thick enzymatic property of M-Zyme, have studied operative temperature (30,40,45,50,55,60,70 and 80 DEG C) and action pH (3.0-10.0) respectively to the impact of this enzyme activity, find that the optimum temperature of this M-Zyme is 50-60 DEG C, optimal pH is 8.5, and this enzyme enzyme activity stable (± 10%) in alkalescence (pH7.0-10.0) environment.This enzyme is middle temperature basic keratins enzyme.
Claims (2)
1. a strain M-Zyme producing bacterial strain streptomyces aureus (
streptomyces aureofaciens), called after K13, be now preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No.8047, and preservation date is on August 19th, 2013.
2. the cultural method of the bacterial strain of preserving number CGMCC No.8047, its substratum is: wool 0.5-1%, glucose 4-8%, peptone 3-5%, SODIUMNITRATE 0.3%, dipotassium hydrogen phosphate 0.04%, sodium-chlor 0.05%, shaking table culture condition is: initial pH value is 10.0, inoculum size 10%, culture temperature 30 DEG C, rotating speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h.
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| CN103642739B true CN103642739B (en) | 2015-09-30 |
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| 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 |
| CN105112344A (en) * | 2015-10-08 | 2015-12-02 | 江南大学 | Brevibacillus parabrevis producing keratinase and application thereof |
| CN110317748B (en) * | 2019-06-06 | 2022-05-24 | 华南理工大学 | Streptomyces strain and application thereof in feather degradation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800358A (en) * | 2005-12-27 | 2006-07-12 | 云南师范大学 | Keratinase-proudicng bacterium and its preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800358A (en) * | 2005-12-27 | 2006-07-12 | 云南师范大学 | Keratinase-proudicng bacterium and its preparation method |
Non-Patent Citations (3)
| Title |
|---|
| 1株角蛋白酶产生菌的筛选、鉴定与产酶特性研究;张竹慧 等;《江苏农业科学》;20130325;第41卷(第3期);315-318 * |
| Biochemical features of microbial keratinases and their production and applications;Brandelli A et al;《Appl Microbiol Biotechnol》;20091229;第85卷(第6期);1735-1750 * |
| Purification and characterization of keratinase from a new Bacillus subtilis strain;Cheng-gang Cai et al;《J Zhejiang Univ Sci》;20080930;第9卷(第9期);713-720 * |
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