CN105199983B - The application of one bacillus amyloliquefaciens and its protease of generation in terms of process hides depilation - Google Patents
The application of one bacillus amyloliquefaciens and its protease of generation in terms of process hides depilation Download PDFInfo
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- CN105199983B CN105199983B CN201510609764.5A CN201510609764A CN105199983B CN 105199983 B CN105199983 B CN 105199983B CN 201510609764 A CN201510609764 A CN 201510609764A CN 105199983 B CN105199983 B CN 105199983B
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Abstract
The invention discloses a kind of bacillus amyloliquefaciens, which is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 14th, 2015, and preserving number is CGMCC No.11067.The invention also discloses a kind of preparation methods of bacillus amyloliquefaciens, and the invention also discloses the protease that a kind of bacillus amyloliquefaciens generate, and the invention also discloses a kind of application of bacillus amyloliquefaciens in terms of process hides depilation.The protease that bacillus amyloliquefaciens of the present invention generate all has preferable depilatory active to the common sheepskin of process hides, pigskin and ox-hide.It is wherein used for ox-hide and the hair removal effect of sheepskin is best, the pelt after depilation is soft, has bating effect, and grain is smooth, pore is clear.
Description
Technical field
The invention belongs to technical field of microbiology, and in particular to a bacillus amyloliquefaciens, the invention further relates to one
The preparation method of kind bacillus amyloliquefaciens, the invention further relates to the protease that a kind of bacillus amyloliquefaciens generate, the present invention
Further relate to a kind of application of bacillus amyloliquefaciens in terms of process hides depilation.
Background technology
Leather industry is listed in the pollution industry for being only second to paper industry.Used in traditional depilation operation vulcanized sodium,
NaHS takes hair-destroying process to lose hair or feathers, and not only generates S2-Pollution, and COD, BOD value in waste water is made to increase, waste water is in highly basic
Property and carry apparent foul smell, serious pollution is caused to environment.As some middle-size and small-size tanneries are strangled successively in recent years
It enables and stops production or limit the production, the survival and development of leather industry are faced with severe tests.Leather industry must realize cleanly production, and
And must veritably implement in leather-making enterprises, the difficult situation that current leather industry is faced could be reversed, realizes leather industry
Sustainable development.
It is to realize one of the effective way of cleaner leather-making production to carry out depilation with biological agent enzyme substitution vulcanized sodium.Angle egg
Keratin degrading is polypeptide and amino acid, therefore can make because the collenchyme in skin can be hydrolyzed preferably by white enzyme
For process hides depilatory agent or auxiliary agent.
Keratinase is found there is apparent depilation ability by Hublin in 1999 et al. for ox-hide depilation research,
Hair degradations of the J.Friedrich et al. by keratinase for pigskin, ox-hide, sheepskin, people is studied between 2003-2005, together
When with collagen be substrate specificity, it is found that keratinase regardless of solution collagen, solves the problems, such as that enzymatic depilation does not destroy collagen.External
Some research reports show that in the environment of not sulfide, the depilation ability of keratinase is very strong.Using containing keratinase
Depilation auxiliary agent, it is possible to reduce TDS and BOD contents in depilation waste liquor, and can largely reduce the dosage of sulfide so that reach
To small incidental expenses amount, this sustainable development and environment friendly important in inhibiting to leather industry.Therefore keratinase is in process hides
Application prospect in depilation is good.
Invention content
The object of the present invention is to provide a kind of bacillus amyloliquefaciens.
It is a further object of the present invention to provide a kind of preparation methods of bacillus amyloliquefaciens.
It is a further object of the present invention to provide the protease that a kind of bacillus amyloliquefaciens generate.
It is a further object of the present invention to provide a kind of application of bacillus amyloliquefaciens in terms of process hides depilation.
First technical solution of the present invention is a bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens), which it is general on July 14th, 2015 to be preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, preserving number are CGMCC No.11067.
Second technical solution of the present invention is a kind of preparation method of bacillus amyloliquefaciens, including following step
Suddenly：
Step 1, sample collection：The soil of putrefying animal hair is carried at tannery's sewer outlet；
Step 2, enrichment：It will sample in access enriched medium and cultivate 48h, be transferred to liquid seed culture medium；
The primary dcreening operation of step 3, bacterial strain：After liquid seed culture medium culture 12h, by bacteria suspension on casein Selective agar medium
For 24 hours, picking has the uniform dibbling of single bacterium colony that hydrolysis is enclosed in casein Selective agar medium for scribing line culture, 37 DEG C of constant temperature incubations 48 hours,
It observes bacterium colony upgrowth situation and hydrolyzes situation with casein；
Step 4, the secondary screening of bacterial strain：Picking hydrolysis encloses the apparent uniform dibbling of bacterium colony on solid wool culture medium, 37 DEG C
Constant temperature incubation 24 hours or more, observation bacterium colony upgrowth situation hydrolyze situation with casein, and then picking hydrolysis circle is at 1 centimetre or more
Single bacterium colony activates 12 hours in seed culture medium, and bacterium solution is measured in fermentation medium by 6% inoculation, 37 DEG C of shaking table hairs
Ferment 36 hours, zymotic fluid is centrifuged；
Step 5,16sDNA sequences：16SrDNA sequences, sequence such as SEQ are carried out to the K20 bacterium filtered out
Shown in ID NO.1, bacillus amyloliquefaciens are prepared.
Further, the component and content of enriched medium are as follows：Peptone:1.0%, beef extract:1.0%, NaCl:
0.5%, K2HPO4:0.07%, casein:1.0%.
Further, primary dcreening operation culture medium is liquid seed culture medium, and component and content are as follows：Glucose：1.5%,
K2HPO4：0.07%, asparagine：0.09%, beef extract：0.07%.
Further, the component and content of casein Selective agar medium are as follows：Glucose：1.5%, K2HPO4：0.07%, day
Winter amide：0.09%, beef extract：0.07%, casein：2.0%, agar：5.0%.
Further, the component and content of solid wool culture medium are as follows：Glucose 1.5%, beef extract 0.1%, asparagus fern
Amide 0.11%, K2HPO40.12%, wool 0.2%, agar：5%, pH6.4.
Further, the component and content of fermentation medium are as follows：Starch：3.0%, K2HPO4：0.12%, asparagine：
0.11%, beef extract：0.05%, wool：0.2%, pH6.4.
Third technical solution of the present invention is that a kind of protease that above-mentioned bacillus amyloliquefaciens generate leads to
Following methods are crossed to be prepared：Bacillus amyloliquefaciens were fermented with fermentation medium by 72 hours, and what is obtained sends out containing protease
Zymotic fluid.
Further, the component and content of fermentation medium are as follows：Starch：3.0%, K2HPO4：0.12%, asparagine：
0.11%, beef extract：0.05%, wool：0.2%, pH6.4.
4th technical solution of the present invention is that a kind of protease by above-mentioned bacillus amyloliquefaciens generation exists
Application in terms of process hides depilation.
The beneficial effects of the invention are as follows：
(1) primary dcreening operation obtains 37 plants of bacterium, 15 plants of bacterium with hydrolysis circle is picked out, by casein selectivity tablet and fermentation
Liquid enzyme activity determination secondary screening has obtained one plant of higher bacterial strain of keratinase vigor, has been named as K20.
(2) it is found by strain morphologic observation:The bacterial strain is that round, flat, diameter is about 2mm, is creamy white, and surface is dry
Dry smooth, colony edge is in patellate.The bacterium is in rod-shaped, through proving that Gram's staining is the positive repeatedly.It is raw under aerobic conditions
It is long good, it is aerobic bacteria.
(3) aerobic growth of K20 bacterium, gelatin hydrolysis, catalase, tyrosine hydrolysis, Starch Hydrolysis, nitrate reduction, V-P
Reaction is the positive；Phenylalanine dehydrogenase, citrate are utilized as feminine gender；Using D-Glucose, D-MANNOSE and malt
Sugar.It is grown at 30 DEG C of pH6-7,2%NaCl, 5 DEG C and 50 DEG C of temperature is not grown.5%NaCl is not grown.In conjunction with the form of bacterial strain
Feature and coloration result can judge K20 bacterium for bacillus or bacillus genus substantially.
(4) judge the bacterial strain for bacillus (Bacillus) through l6s rDNA sequence analyses.Through base sequence
NCBI is compared, and can tentatively assert that the bacterial strain is bacillus amyloliquefaciens.
(5) protease that K20 bacterium generate all has preferable depilatory active to the common sheepskin of process hides, pigskin and ox-hide.
It is wherein used for ox-hide and the hair removal effect of sheepskin is best, the pelt after depilation is soft, has bating effect, and grain is smooth, pore
Clearly.
Description of the drawings
K1~K20 bacterium colonies upgrowth situation when Fig. 1 is primary dcreening operation of the present invention；
K21~K37 bacterium colonies upgrowth situation when Fig. 2 is primary dcreening operation of the present invention；
Fig. 3 is growth (K5, K10, K15, K20, K21, K22 bacterial strain) of the bacterium colony of the present invention on solid medium；
Fig. 4 is K20 bacterial strains colony morphology characteristic figure of the present invention；
Fig. 5 is Gram's staining microphoto X100 of the present invention；
Fig. 6 is PCR amplification 16s rDNA results of the present invention；
Fig. 7 is the knot that the protease that the bacterial strain that the present invention is prepared generates carries out Sichuan Native Breed of Goats hair depilation processing
Fruit；
Fig. 8 is the result that the protease that the bacterial strain that the present invention is prepared generates carries out French milk ox-hide depilation processing；
The protease that the bacterial strain that Fig. 9 present invention is prepared generates carries out Australia oxhide the result of depilation processing.
Specific implementation mode
The present invention is described in detail With reference to embodiment.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
1. materials and methods
1.1. material
1.1.1 laboratory apparatus
1.1.2 experiment reagent
1.1.3 enriched medium
Enriched medium (%)
Peptone:1.0, beef extract:1.0, NaCl:0.5, K2HPO4:0.07, casein:1.0；
1.1.4 primary dcreening operation culture medium
1. liquid seed culture medium (%)
Glucose：1.5, K2HPO4：0.07, asparagine：0.09, beef extract：0.07；
2. casein Selective agar medium (%)
Glucose：1.5, K2HPO4：0.07, asparagine：0.09, beef extract：0.07, casein：2.0, agar：5.0；
1.1.5 secondary screening culture medium：
1. solid wool culture medium (%)
Glucose 1.5, beef extract 0.1, asparagine 0.11, K2HPO40.12,0.2 agar of wool：5pH6.4；
2. fermentation medium (%)
Starch：3.0, K2HPO4：0.12, asparagine：0.11, beef extract：0.05, wool：0.2pH6.4.
The separation and identification of 1 bacterium of embodiment
1.1. sample collection：The soil of putrefying animal hair is carried at tannery's sewer outlet.
1.2. enrichment：It will sample in access enriched medium and cultivate 48h, be transferred to liquid seed culture medium.
1.3. the primary dcreening operation of bacterial strain
After liquid seed culture medium culture 12h, bacteria suspension is crossed into culture for 24 hours on casein Selective agar medium, picking
There is the uniform dibbling of single bacterium colony that hydrolysis is enclosed in casein Selective agar medium.Bacterium colony number is № 1-37,37 DEG C of constant temperature incubations 48 hours,
It observes bacterium colony upgrowth situation and hydrolyzes situation with casein.
1.4. the secondary screening of bacterial strain
Picking hydrolysis encloses the apparent uniform dibbling of bacterium colony on solid wool culture medium, 37 DEG C of constant temperature incubations 24 hours with
On, observation bacterium colony upgrowth situation hydrolyzes situation with casein.Then single bacterium colony of the picking hydrolysis circle at 1 centimetre or more is trained in seed
It supports and activate 12 hours in base, by 6% inoculation measurement bacterium solution in fermentation medium, 37 DEG C of shaker fermentations 36 hours.It will fermentation
Liquid centrifuges, and the stillness of night is taken to use folin's methods to measure fermentation broth enzyme activity with wool method respectively.
1.5. folin's methods
2mL2% caseins substrate is taken (to prepare, pH value 8.0) solution (blank cuvette with potassium dihydrogen phosphate borax buffer solution
Middle addition solution of trichloroacetic acid), it is placed in (40 ± 0.2) DEG C water-bath preheating, 1mL enzyme solutions is separately added into and shakes up.From starting plus enzyme solution
Timing is played, solution of trichloroacetic acid is added after ten minutes and terminates reaction (in blank cuvette plus casein solution), centrifugation takes supernatant
Colour developing measures absorbance value at 560nm.
Enzyme activity defines：The enzyme amount that 1min is catalytically decomposed out needed for 1 μ g tyrosine in above-mentioned reaction system is defined as one
A enzyme activity unit (U/m L).
Enzyme activity U/ml=OD value * K*6*n/10
OD values：Absorbance value
K：According to the calculated slope of tyrosine standard curve.
n：The extension rate of enzyme solution
1.6. wool method
Under the conditions of pH9.0 and 40 DEG C, the suitably diluted enzyme solutions of 1ml are taken, 1% soluble keratins of 2ml, control is added
Only add the enzyme solution of 1ml, 55 DEG C of water-bath 15min.After reaction, 1ml TCA (trichloroacetic acid) are added and terminate reaction, control group
1% soluble keratins of 2ml are added again, overturn mixing, stand 5min, and 5min is centrifuged in 10000rpm.Take the supernatant after centrifugation
1ml sodium carbonate is added in liquid 1ml, and 1ml forint phenol Folin reagents (every group of three parallel laboratory tests) mixing develops the color in 40 DEG C of water-baths
20min.It is that 680nm measures OD values in wavelength,
Keratinase can be by keratin hydrolysis at the even free amino acid of small peptide, and enzyme activity is higher, the amino acid of generation
It is more.The OD values of reaction solution are measured in 750nm.
Enzyme activity defines：It is an enzyme activity list that absorbance value, which often increases by 0.01, in above-mentioned reaction system, under wavelength 680nm
Position (U/mL).If being converted into specific enzyme activity (U/g or U/mg), need divided by enzyme solution concentration.
Enzyme activity U/ml=OD value * K*6*n/10
OD values：Absorbance value
K：According to the calculated slope of tyrosine standard curve.
n：The extension rate of enzyme solution
1.7. colony morphological observation
Conventional method makes bacteria suspension smear, observes colonial morphology under the microscope.
Gram's staining is carried out to the K20 bacterium filtered out.
1.8. 16sDNA sequences
16SrDNA sequences are carried out to the K20 bacterium filtered out.
1.9.K20 the keratinase depilation performance pre-test that bacterium generates
Enzyme solution is applied to through centrifuging and taking supernatant by the dosage of 200U/g sheepskins and 250U/g ox-hides by K20 fermented liquids
The flesh noodles layer of skin carries out banking up enzymatic depilation at 35 DEG C.Regular check depilation situation.As a result it see the table below.
1.10 experimental result：
1.10.1 primary dcreening operation result
1.10.1.1 tablet primary dcreening operation
As shown in Figure 1, Figure 2 and Figure 3, it is observed that K5, K10, K15, K20, K21, K22, K23, K24, K25, K26, K28,
15 plants of bacterium of K29, K30, K31, K33 have hydrolysis to enclose, and will be cultivated on this 15 plants of bacterium again dibbling to casein selective medium,
It was found that hydrolysis circle has six plants of bacterium at 1 centimetre or more, as shown in Figure 3.The hydrolysis circle of wherein K20 bacterium is maximum.To this six plants of bacterium into
Row fermentation secondary screening.
1.10.1.2 secondary screening result
It is measured by six plants of fermented liquid keratinase vigor to filtering out, acquired results are shown in Table 1.
The keratinase vigor and prolease activity that 1 bacterial strain of table generates
The proteolysis enzyme activity and keratinase vigor of bacterial strain fermentation liquor measure display, and K20 bacterial strains have protease activity concurrently
Property and keratinase activity, prolease activity it is higher than other bacterial strains, it is observed when the enzyme activity measured is with tablet culture
Larger hydrolysis circle matches (shown in Fig. 3).Therefore it is for further study K20 bacterium to be selected.
2 K20 bacterial strain physiological biochemical properties of embodiment are studied
2.1. morphological feature and coloration result
K20 bacterial strain colony morphology characteristics:Shape:Circle, it is flat.Diameter about 2mm.Milky, dry tack free is smooth, bacterium colony
Edge is in patellate.See Fig. 4.
It is in rod-shaped that K20 bacterial strains are observed under the microscope, through proving that Gram's staining is positive (see Fig. 5) repeatedly.Aerobic
Under the conditions of well-grown, be aerobic bacteria.Gemma is produced, gemma is ellipse, there is motility.
2.2 physiological and biochemical property testing results
The gelatin hydrolysis of K20 bacterium, aerobic growth, catalase, tyrosine hydrolysis, Starch Hydrolysis, nitrate reduction, V-P are anti-
It should be the positive；Phenylalanine dehydrogenase, citrate are utilized as feminine gender；Using D-Glucose, D-MANNOSE and maltose.
It is grown under 30 DEG C, pH6-7,2%NaCl, 5 DEG C and 50 DEG C of temperature is not grown.5%NaCl is not grown.Form in conjunction with bacterial strain is special
Sign and coloration result, can judge K20 bacterium for bacillus or bacillus genus substantially.
The physiological and biochemical property of 2 K20 bacterium of table
2.3 16s rDNA sequence analyses
Utilize universal primer (the 5 '-AGAGTTTGATCCTGGCTCAG- of sense primer of amplification bacterium 16s rDNA genes
3 ', downstream primer 5 '-CGGTTACCTTGTTACGACTT-3 '), the 16S for obtaining being about 1.5kb is expanded from bacterial strain total DNA
RDNA (rRNA) genetic fragment (see Fig. 6), has carried out nucleotide sequencing, and the sequence measured, will as shown in SEQ ID NO.1
The sequence is compared (Fig. 4) with the 16S rDNA sequences of GenBank databases, is found with bacterial strain 16S rDNA homologys most
High is the 16S rDNA sequences of bacillus (Bacillus), has 100% homology with multiple sequences.Therefore, may be used
The preliminary bacterial strain of the bacterial strain is accredited as bacillus.
Known to the NCBI comparing results of above-mentioned base sequence：The genetic evolution distance of K20 bacterial strains and bacillus are nearest,
It belongs to a minimum branch with bacillus amyloliquefaciens.Morphological feature in conjunction with K20 bacterial strains and 16S rDNA sequence analyses,
Identify that K20 bacterial strains are bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The protease depilation performance test result that 3 K20 bacterium of embodiment generate
K20 bacterium were fermented with 2. fermentation medium (%) by 72 hours, and what is obtained is used for doing losing hair or feathers containing protease fermented liquid
Experiment.Wherein, 2. the component and content of fermentation medium (%) are as follows：Starch：3.0, K2HPO4：0.12, asparagine：0.11,
Beef extract：0.05, wool：0.2pH6.4.
The experimental results showed that：The protease that K20 bacterium generate all has the common sheepskin of process hides, pigskin and ox-hide preferable
Depilatory active (table 3)：It is wherein used for ox-hide and the hair removal effect of sheepskin is best.Pelt after depilation is soft, there is bating effect, and
Grain is smooth, and pore is clear.
The protease depilation activity that 3 bacterial strain of table generates
+ hair removal effect is evaluated ,+more, hair removal effect is better
The protease generated using the bacterial strain that the present invention is prepared is to Sichuan Native Breed of Goats hair, French milk ox-hide and Australia
Continent oxhide carries out depilation processing, and the pelt after depilation is soft, has bating effect, and grain is smooth, the clear (such as Fig. 7-figure of pore
Shown in 9).
Claims (4)
1. a bacillus amyloliquefaciens, which is characterized in that the bacterial strain was preserved in Chinese microorganism strain on July 14th, 2015
Preservation administration committee common micro-organisms center, preserving number are CGMCC No.11067.
2. a kind of protease generated by bacillus amyloliquefaciens described in claim 1, which is characterized in that by the following method
It is prepared：Bacillus amyloliquefaciens were fermented with fermentation medium by 72 hours, and what is obtained contains protease fermented liquid.
3. the protease that bacillus amyloliquefaciens according to claim 2 generate, which is characterized in that the fermentation medium
Component and content it is as follows：Starch：3.0%, K2HPO4：0.12%, asparagine：0.11%, beef extract：0.05%, wool：
0.2%, pH6.4.
4. a kind of application of the protease that bacillus amyloliquefaciens by described in claim 2 generate in terms of process hides depilation.
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| CN106167816B (en) * | 2016-07-06 | 2019-10-01 | 贵州大学 | The method for improving porcine haemoglobin degree of hydrolysis with bacillus amyloliquefaciens GUHP86 |
| CN107699553B (en) * | 2017-11-03 | 2021-04-09 | 天津科技大学 | Alkaline keratinase KerT from bacillus amyloliquefaciens and unhairing application thereof |
| CN111996154A (en) * | 2020-09-24 | 2020-11-27 | 上海市环境监测中心(上海长三角区域空气质量预测预报中心) | Bacillus subtilis black variant culture medium and preparation method and application thereof |
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