CN103642739A

CN103642739A - Streptomces aureofaciens for producing keratinase and application method thereof

Info

Publication number: CN103642739A
Application number: CN201310701537.6A
Authority: CN
Inventors: 张旦旦; 王月; 史劲松; 许正宏; 张晓梅
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2013-12-19
Filing date: 2013-12-19
Publication date: 2014-03-19
Anticipated expiration: 2033-12-19
Also published as: CN103642739B

Abstract

The invention discloses a keratin producing bacterial strain as well as culture conditions and crude enzyme property thereof and belongs to the technical field of microorganisms. Through identification, the bacterial strain is Streptomyces aureofaciens, named as K13 and preserved in CGMCC (China General Microbiological Culture Collection Center) with the preservation number of CGMCC NO.8047. Through preliminary optimization, the culture medium of the bacterial strain comprises 0.5-1% of wool, 4-8% of glucose, 3-5% of peptone, 0.3% of sodium nitrate, 0.04% of dipotassium phosphate and 0.05% of sodium chloride; the shake culture conditions are that the initial pH (potential of Hydrogen) value is 10.0, the inoculation quantity is 10%, the culture temperature is 30 DEG C, the rotating speed is 220 revolutions per minute, the liquid loading volume is 30ml/250ml, the fermentation period is 64 hours and the keratinase producing activity is up to 44.2U/ml. The keratinase is mesophilic alkaline keratinase, the optimum operation temperature is 50-60 DEG C and the optimum pH is 8.5; the enzyme activity of the keratinase is stable in an alkaline environment (pH 7.0-10.0).

Description

Streptomyces aureus and the application method thereof of M-Zyme produced in one strain

Technical field

The invention belongs to microbial technology field, be specifically related to streptomyces aureus and thick enzymatic property thereof that M-Zyme is produced in a strain.

Background technology

Annual a large amount of poultry feathers and the useless time wool of producing of China, 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 common protease hydrolysis, thereby major part is not utilized, 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, and reduces environmental pollution.Many microorganisms can produce M-Zyme, as streptomycete, Zymomonas mobilis, genus bacillus etc.

The object of the present invention is to provide a strain to produce the streptomyces aureus of M-Zyme, and preliminary study its thick enzymatic property.

Summary of the invention

the methods such as the screening of M-Zyme producing bacterial strain, evaluation, culture condition optimization are as follows:

(1) dull and stereotyped primary dcreening operation: gather pedotheque near rural area sheepfold Southern Yangtze University, get 1g soil sample and add in 10ml physiological saline, prepare soil supension, dilution spread, to primary dcreening operation substratum plate, is cultivated 3d for 30 ℃.Larger single bacterium colony that picking can be grown 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, is measured its supernatant liquor M-Zyme vigor, finds that No. 13 bacterium enzyme activities are higher.

(3) bacterial strain 16S rDNA identifies: extract total DNA, with bacterial 16 S rDNA universal primer, increase, the gene order obtaining is 1500 bp, through order-checking comparison, for streptomyces aureus (Streptomyces aureofaciens), called after K13, is deposited in the China Committee for Culture Collection of Microorganisms's common micro-organisms center that is 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 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%, 30 ℃ of culture temperature, shaking speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h, produces M-Zyme vigor and reaches 44.2U/ml.

(5) thick Characterizaton: studied respectively the impact on this enzyme activity of operative temperature and action pH, the optimum temperature of finding this M-Zyme is 50-60 ℃, optimal pH is 8.5, and this enzyme enzyme activity stable (± 10%) in alkalescence (pH7.0-10.0) environment.This enzyme is the alkaline M-Zyme of middle temperature.

(6) measuring method of M-Zyme vigor: get a 18ml test tube, the pH Tris-HCl damping fluid 2.0ml, the wool powder substrate 10mg that add successively crude enzyme liquid (fermented supernatant fluid) 1.0ml, 0.05mol/l, 37 ℃ of constant temperature oscillation water-bath reaction 1h, the trichoroacetic acid(TCA) 2.0mL termination reaction that adds 0.4mol/l, centrifuging and taking supernatant liquor is measured its light absorption value in 280nm.To add the reaction tubes of trichoroacetic acid(TCA) before 37 ℃ of constant temperature oscillations, compare.

Enzyme activity definition: 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 waters.

Embodiment

screening and the evaluation 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, and 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

From near rural area sheepfold Southern Yangtze University, gather pedotheque, get in the 50 ml triangular flasks that 1 g soil sample adds 10 ml physiological saline and several granulated glass spherees to, shaking table vibrates 15 min after soil sample is disperseed, standing 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, make thus each extent of dilution soil supension.(placement is spent the night to primary dcreening operation culture medium flat plate to draw respectively each extent of dilution suspension 0.1 ml, without varied bacteria growing), in 30 ℃ of constant temperature culture 3d, with the transfering loop bacterium colony coming 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, selects the better bacterial strain of growth to continue 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, adopt respectively Gao Shi substratum, LB substratum and czapek's solution to cultivate activation preparation seed liquor, inoculum size by 2% is seeded to fermention medium, 30 ℃, 220 r/min cultivate 64 h, measuring the vigor of fermented supernatant fluid M-Zyme, find that No. 13 bacterium enzyme activities are higher, is 3.9 U/ml.

4,16S rDNA identifies

Extract total DNA, with the amplification of bacterial 16 S rDNA universal primer, the gene order obtaining is 1500 bp, through order-checking comparison, with the 16S rDNA sequence homology of streptomyces aureus up to 99%, tentatively judge its be streptomyces aureus ( streptomyces aureofaciens), called after K13.

Embodiment 2 strain fermentation condition initial optimizations

Utilize single factor 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%, 30 ℃ of culture temperature, shaking speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h.It produces M-Zyme vigor and reaches 44.2U/ml, before optimizing, has improved 10.3 times.

the thick Characterizaton of embodiment 3

Preliminary study the thick enzymatic property of the M-Zyme that produces, operative temperature (30,40,45,50,55,60,70 and 80 ℃) and the impact of action pH (3.0-10.0) on this enzyme activity have been studied respectively, the optimum temperature of finding this M-Zyme is 50-60 ℃, optimal pH is 8.5, and this enzyme enzyme activity stable (± 10%) in alkalescence (pH7.0-10.0) environment.This enzyme is the alkaline M-Zyme of middle temperature.

Claims

One strain M-Zyme producing bacterial strain streptomyces aureus ( streptomyces aureofaciens), called after K13, is now preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.8047, and preservation date is on August 19th, 2013.
2. the cultural method of bacterial strain described in claim 1, 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%, 30 ℃ of culture temperature, rotating speed 220r/min, liquid amount 30ml/250ml, fermentation period 64h, produces M-Zyme vigor and reaches 44.2U/ml.
3. the M-Zyme that described in claim 1, bacterial strain produces is the alkaline M-Zyme of middle temperature, and optimum temperature is 50-60 ℃, and optimal pH is 8.5, and this enzyme enzyme activity in alkalescence (pH7.0-10.0) environment is stable.