CN105199983A - Bacillus amyloliquefaciens and application of protease produced by same in tanning depilation - Google Patents
Bacillus amyloliquefaciens and application of protease produced by same in tanning depilation Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens. The strain is preserved in the China General Microbiological Culture Collection Center on July 14th, 2015, and the preservation number is CGMCC No.11067. The invention further discloses a preparation method of the bacillus amyloliquefaciens. The invention further discloses protease produced by the bacillus amyloliquefaciens and further discloses an application of the bacillus amyloliquefaciens in tanning depilation. The protease produced by the bacillus amyloliquefaciens has good depilation activity in sheepskin, pigskin and cow leather which are frequently used in tanning, wherein the depilation effects on the cow leather and the sheepskin are the best, naked leather which is subjected to the depilation is soft, a softening effect is achieved, the grain surface is smooth, and pores are clear.
Description
Technical field
The invention belongs to technical field of microbiology, be specifically related to a bacillus amyloliquefaciens, the invention still further relates to a kind of preparation method of bacillus amyloliquefaciens, the invention still further relates to the proteolytic enzyme that a kind of bacillus amyloliquefaciens produces, the invention still further relates to the application of a kind of bacillus amyloliquefaciens in process hides depilation.
Background technology
Leather industry is listed in the pollution industry being only second to paper industry.Employ sodium sulphite, Sodium sulfhydrate in traditional depilation operation, take hair-destroying process to lose hair or feathers, not only produce S
2-pollute, and COD, BOD value in waste water is raised, waste water is strong basicity and with obvious foul smell, causes serious pollution to environment.Along with some middle-size and small-size tanneries are ordered stopping production or limited production successively in recent years, the survival and development of leather industry are faced with severe tests.Leather industry must realize cleanly production, and must implement at leather-making enterprises veritably, could reverse the difficult situation that current leather industry faces, realize the Sustainable development of leather industry.
Carrying out losing hair or feathers with biotechnological formulation enzyme replacement sodium sulphite is one of effective way realizing cleaner leather-making production.Keratin degrading, because can be hydrolyzed the collenchyme in skin preferably, is polypeptide and amino acid by M-Zyme, therefore can as process hides trichogen or auxiliary agent.
M-Zyme is used for ox-hide depilation research discovery and has obvious depilation ability by the people such as Hublin in 1999, between 2003-2005, M-Zyme is used for the hair degradation research of pigskin, ox-hide, sheepskin, people by the people such as J.Friedrich, be substrate specificity with collagen simultaneously, find M-Zyme not decomposes collagen, solve the difficult problem that enzyme unhairing does not destroy collagen.Some external research reports show, in the environment not having sulfide, the depilation ability of M-Zyme is very strong.Use the depilation auxiliary agent containing M-Zyme, can reduce TDS and BOD content in depilation waste liquor, and can reduce the consumption of sulfide in a large number so that reach small incidental expenses amount, this is to the Sustainable development of tanning industry and environment friendly important in inhibiting.Therefore the application prospect of M-Zyme in process hides depilation is had an optimistic view of.
Summary of the invention
The object of this invention is to provide a kind of bacillus amyloliquefaciens.
Another object of the present invention is to provide a kind of preparation method of bacillus amyloliquefaciens.
Another object of the present invention is to provide the proteolytic enzyme that a kind of bacillus amyloliquefaciens produces.
Another object of the present invention is to provide the application of a kind of bacillus amyloliquefaciens in process hides depilation.
First technical scheme of the present invention is, one bacillus amyloliquefaciens (Bacillusamyloliquefaciens), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 14th, 2015, and preserving number is CGMCCNo.11067.
Second technical scheme of the present invention is that a kind of preparation method of bacillus amyloliquefaciens, comprises the following steps:
Step 1, sample collecting: sewer outlet place of tannery is with the earth of putrefying animal hair;
Step 2, enrichment: by sample access enrichment medium in cultivate 48h, proceed to liquid seed culture medium;
The primary dcreening operation of step 3, bacterial strain: after liquid seed culture medium cultivates 12h, by bacteria suspension streak culture 24h on casein Selective agar medium, picking has the even dibbling of single bacterium colony of hydrolysis circle in casein Selective agar medium, and 37 DEG C of constant temperature culture 48 hours, observe colony growth situation and casein is hydrolyzed situation;
The multiple sieve of step 4, bacterial strain: the even dibbling of the picking hydrolysis obvious bacterium colony of circle is on solid wool substratum, 37 DEG C of constant temperature culture more than 24 hours, observation colony growth situation and casein are hydrolyzed situation, then the single bacterium colony of picking hydrolysis circle more than 1 centimetre, 12 hours are activated in seed culture medium, inoculation by 6% measures bacterium liquid in fermention medium, 37 DEG C of shaker fermentations 36 hours, and fermented liquid is centrifugal;
Step 5,16sDNA sequence: carry out 16SrDNA sequence to the K20 bacterium filtered out, its sequence, as shown in SEQIDNO.1, prepares bacillus amyloliquefaciens.
Further, component and the content of enrichment medium are as follows: peptone: 1.0%, extractum carnis: 1.0%, NaCl:0.5%, K
2hPO
4: 0.07%, casein: 1.0%.
Further, primary dcreening operation substratum is liquid seed culture medium, its component and content as follows: glucose: 1.5%, K
2hPO
4: 0.07%, l-asparagine: 0.09%, extractum carnis: 0.07%.
Further, the component of casein Selective agar medium and content as follows: glucose: 1.5%, K
2hPO
4: 0.07%, l-asparagine: 0.09%, extractum carnis: 0.07%, casein food grade: 2.0%, agar: 5.0%.
Further, component and the content of solid wool substratum are as follows: glucose 1.5%, extractum carnis 0.1%, l-asparagine 0.11%, K
2hPO
40.12%, wool 0.2%, agar: 5%, pH6.4.
Further, the component of fermention medium and content as follows: starch: 3.0%, K
2hPO
4: 0.12%, l-asparagine: 0.11%, extractum carnis: 0.05%, wool: 0.2%, pH6.4.
3rd technical scheme of the present invention is, the proteolytic enzyme that a kind of above-mentioned bacillus amyloliquefaciens produces, and prepares by the following method: bacillus amyloliquefaciens fermention medium is through 72 hours fermentation, and what obtain contains protease fermented liquid.
Further, the component of fermention medium and content as follows: starch: 3.0%, K
2hPO
4: 0.12%, l-asparagine: 0.11%, extractum carnis: 0.05%, wool: 0.2%, pH6.4.
4th technical scheme of the present invention is, a kind of application of proteolytic enzyme in process hides depilation produced by above-mentioned bacillus amyloliquefaciens.
The invention has the beneficial effects as follows:
(1) primary dcreening operation obtains 37 strain bacterium, picks out the bacterium that 15 strains have hydrolysis circle, sieves again, obtain the bacterial strain that a strain M-Zyme vigor is higher, called after K20 through casein selectivity flat board and fermentation broth enzyme vitality test.
(2) found by bacterial classification morphologic observation: this bacterial strain is about 2mm for circular, smooth, diameter, and be creamy white, surface drying is smooth, and colony edge is patellate.This bacterium is shaft-like, through repeatedly proving that gramstaining is positive.Well-grown under aerobic conditions is aerobic bacteria.
(3) aerobic growth of K20 bacterium,
gelatinhydrolysis, catalase, tyrosine hydrolysis, Starch Hydrolysis, nitrate reduction, V-P reaction is the positive; Phenylalanine dehydrogenase, Citrate trianion is utilized as feminine gender; D-Glucose can be utilized, D-MANNOSE and maltose.Grow under 30 DEG C of pH6-7,2%NaCl, temperature 5 DEG C and 50 DEG C do not grow.5%NaCl does not grow.In conjunction with morphological specificity and the coloration result of bacterial strain, substantially can judge that K20 bacterium is that genus bacillus or series bacillus belong to.
(4) judge that this bacterial strain is bacillus (Bacillus) through l6srDNA sequential analysis.Contrast through the NCBI of base sequence, tentatively can assert that this bacterial strain is bacillus amyloliquefaciens.
(5) proteolytic enzyme that K20 bacterium produces all has good depilatory active to sheepskin, pigskin and the ox-hide that process hides is conventional.Hair removal effect wherein for ox-hide and sheepskin is best, and the pelt after depilation is soft, has bating effect, and grain is level and smooth, and pore is clear.
Accompanying drawing explanation
fig. 1k1 ~ K20 colony growth situation when being primary dcreening operation of the present invention;
fig. 2k21 ~ K37 colony growth situation when being primary dcreening operation of the present invention;
fig. 3the growth of bacterium colony of the present invention on solid medium (K5, K10, K15, K20, K21, K22 bacterial strain);
fig. 4it is K20 bacterial strain colony morphology characteristic of the present invention
figure;
fig. 5gramstaining Photomicrograph X100 of the present invention;
fig. 6it is pcr amplification 16srDNA result of the present invention;
fig. 7the result of process that to be the proteolytic enzyme that produces of the bacterial strain for preparing of the present invention to Sichuan Native Breed of Goats hair carry out losing hair or feathers;
fig. 8the proteolytic enzyme pair of the bacterial strain generation that the present invention prepares
francemilk ox-hide carries out the result processed of losing hair or feathers;
fig. 9the proteolytic enzyme that the bacterial strain that the present invention prepares produces carries out to Australia oxhide the result processed of losing hair or feathers.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
The experimental technique used in following embodiment if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
1. materials and methods
1.1. material
1.1.1 laboratory apparatus
1.1.2 experiment reagent
1.1.3 enrichment medium
Enrichment medium (%)
Peptone: 1.0, extractum carnis: 1.0, NaCl:0.5, K
2hPO
4:0.07, casein: 1.0;
1.1.4 primary dcreening operation substratum
1. liquid seed culture medium (%)
Glucose: 1.5, K
2hPO
4: 0.07, l-asparagine: 0.09, extractum carnis: 0.07;
2. casein Selective agar medium (%)
Glucose: 1.5, K
2hPO
4: 0.07, l-asparagine: 0.09, extractum carnis: 0.07, casein food grade: 2.0, agar: 5.0;
1.1.5 substratum is sieved again:
1. solid wool substratum (%)
Glucose 1.5, extractum carnis 0.1, l-asparagine 0.11, K
2hPO
40.12, wool 0.2 agar: 5pH6.4;
2. fermention medium (%)
Starch: 3.0, K
2hPO
4: 0.12, l-asparagine: 0.11, extractum carnis: 0.05, wool: 0.2pH6.4.
The Isolation and ldentification of embodiment 1 bacterium
1.1. sample collecting: sewer outlet place of tannery is with the earth of putrefying animal hair.
1.2. enrichment: by sample access enrichment medium in cultivate 48h, proceed to liquid seed culture medium.
1.3. the primary dcreening operation of bacterial strain
After liquid seed culture medium cultivates 12h, by bacteria suspension streak culture 24h on casein Selective agar medium, picking has the even dibbling of single bacterium colony of hydrolysis circle in casein Selective agar medium.Bacterium colony is numbered № 1-37,37 DEG C of constant temperature culture 48 hours, observes colony growth situation and casein is hydrolyzed situation.
1.4. the multiple sieve of bacterial strain
The obvious bacterium colony even dibbling of picking hydrolysis circle is on solid wool substratum, and 37 DEG C of constant temperature culture more than 24 hours, observe colony growth situation and casein is hydrolyzed situation.Then the single bacterium colony of picking hydrolysis circle more than 1 centimetre, activation 12 hours in seed culture medium, the inoculation by 6% measures bacterium liquid in fermention medium, 37 DEG C of shaker fermentations 36 hours.Fermented liquid is centrifugal, get the stillness of night and use folin's methods and wool method to measure fermentation broth enzyme vigor respectively.
1.5. folin's methods
Get 2mL2% casein substrate (with the preparation of potassium primary phosphate borax buffer solution, pH value is 8.0) solution (adding solution of trichloroacetic acid in blank cuvette), be placed in (40 ± 0.2) DEG C water-bath preheating, add 1mL enzyme liquid respectively and shake up.Timing from the enzyme-added liquid of beginning, adds solution of trichloroacetic acid termination reaction (adding casein food grade solution in blank cuvette) after 10 minutes, centrifugal, gets supernatant liquor colour developing, measures absorbance at 560nm place.
Enzyme activity defines: in above-mentioned reaction system, the 1min catalytic decomposition enzyme amount gone out needed for 1 μ g tyrosine is defined as an enzyme activity unit (U/mL).
Enzyme activity U/ml=OD value * K*6*n/10
OD value: absorbance
K: the slope calculated according to tyrosine typical curve.
N: the extension rate of enzyme liquid
1.6. wool method
Under pH9.0 and 40 DEG C condition, get the enzyme liquid that 1ml suitably dilutes, add 2ml1% soluble keratin, the enzyme liquid of 1ml is only added in contrast, 55 DEG C of water-bath 15min.After reaction terminates, add 1mlTCA (trichoroacetic acid(TCA)) termination reaction, control group adds 2ml1% soluble keratin again, puts upside down mixing, leaves standstill 5min, at the centrifugal 5min of 10000rpm.Get centrifugal after supernatant liquor 1ml add 1ml sodium carbonate, 1ml forint phenol Folin reagent (often organize three parallel laboratory tests) mixing, at 40 DEG C of water-baths colour developing 20min.Be that 680nm measures OD value at wavelength,
The amino acid that keratin hydrolysis can become small peptide even to dissociate by M-Zyme, enzyme activity is higher, and the amino acid of generation is more.In the OD value of 750nm assaying reaction liquid.
Enzyme activity defines: in above-mentioned reaction system, under wavelength 680nm, absorbance often increases by 0.01 is a Ge Meihuo unit (U/mL).Live (U/g or U/mg) than enzyme if be converted into, need divided by enzyme liquid concentration.
Enzyme activity U/ml=OD value * K*6*n/10
OD value: absorbance
K: the slope calculated according to tyrosine typical curve.
N: the extension rate of enzyme liquid
1.7. colony morphological observation
Ordinary method makes bacteria suspension smear, examines under a microscope colonial morphology.
Gramstaining is carried out to the K20 bacterium filtered out.
1.8.16sDNA sequence
16SrDNA sequence is carried out to the K20 bacterium filtered out.
1.9.K20 the M-Zyme depilation performance pre-test of bacterium generation
K20 fermented liquid is through centrifuging and taking supernatant liquor, and by the consumption of 200U/g sheepskin and 250U/g ox-hide, flesh noodles layer enzyme liquid being applied to skin carries out banking up enzyme unhairing at 35 DEG C.Quantitative check depilation situation.The results are shown in
following table.
1.10 experimental result:
1.10.1 primary dcreening operation result
1.10.1.1 dull and stereotyped primary dcreening operation
as Fig. 1,
fig. 2with
fig. 3shown in, through observing, K5, K10, K15, K20, K21, K22, K23, K24, K25, K26, K28, K29, K30, K31, K33 15 strain bacterium have hydrolysis circle, this 15 strain bacterium again dibbling is cultivated to casein selective medium, find that hydrolysis circle has six strain bacterium more than 1 centimetre, see
fig. 3shown in.Wherein the hydrolysis circle of K20 bacterium is maximum.This six strains bacterium is fermented multiple sieve.
1.10.1.2 result is sieved again
Through measuring the six strain fermented liquid M-Zyme vigor filtered out, acquired results is shown in
table 1.
table 1the M-Zyme vigor that bacterial strain produces and proteinase activity
The proteolysis enzyme activity of bacterial strain fermentation liquor and M-Zyme vigor measure display, K20 bacterial strain has protease activity and keratinase activity concurrently, its other bacterial strains of protease activity force rate are high, viewed larger hydrolysis circle when the enzyme activity measured and slat chain conveyor (see
fig. 3shown in) match.Therefore K20 bacterium is selected for further study.
Embodiment 2K20 bacterial strain physiological biochemical property is studied
2.1. morphological specificity and coloration result
K20 bacterial strain colony morphology characteristic: shape: circular, smooth.Diameter is about 2mm.Oyster white, surface drying is smooth, and colony edge is patellate.See
fig. 4.
K20 bacterial strain is examined under a microscope in shaft-like, through repeatedly prove gramstaining be positive (see
fig. 5).Well-grown under aerobic conditions is aerobic bacteria.Produce gemma, gemma is oval, has mobility.
2.2 physiological and biochemical property detected results
K20 bacterium
gelatinhydrolysis, aerobic growth, catalase, tyrosine hydrolysis, Starch Hydrolysis, nitrate reduction, V-P reaction is the positive; Phenylalanine dehydrogenase, Citrate trianion is utilized as feminine gender; D-Glucose can be utilized, D-MANNOSE and maltose.At 30 DEG C, grow under pH6-7,2%NaCl, temperature 5 DEG C and 50 DEG C do not grow.5%NaCl does not grow.In conjunction with morphological specificity and the coloration result of bacterial strain, substantially can judge that K20 bacterium is that genus bacillus or series bacillus belong to.
table 2the physiological and biochemical property of K20 bacterium
2.316srDNA sequential analysis
Utilize universal primer (the upstream primer 5 '-AGAGTTTGATCCTGGCTCAG-3 ' of amplification bacterium 16srDNA gene, downstream primer 5 '-CGGTTACCTTGTTACGACTT-3 '), from bacterial strain STb gene amplification obtain being about 1.5kb 16SrDNA (rRNA) gene fragment (see
fig. 6), carried out nucleotide sequencing, the sequence recorded as shown in SEQIDNO.1, the 16SrDNA sequence of this sequence and GenBank database is compared (
fig. 4), find the 16SrDNA sequence that be bacillus (Bacillus) the highest with this bacterial strain 16SrDNA homology, with multiple sequence, there is 100% homology.Therefore, can be genus bacillus by preliminary for this bacterial strain identification of strains.
The NCBI comparing result of above-mentioned base sequence is known: the genetic evolution distance of K20 bacterial strain is nearest with bacillus, and it and bacillus amyloliquefaciens belong to a minimum branch.In conjunction with morphological specificity and the 16SrDNA sequential analysis of K20 bacterial strain, identify that K20 bacterial strain is bacillus amyloliquefaciens (Bacillusamyloliquefaciens).
The protease depilation performance test result that embodiment 3K20 bacterium produces
K20 bacterium is with 2. fermention medium (%) is through 72 hours fermentation, and what obtain is used for doing depilation test containing protease fermented liquid.Wherein, 2. the component of fermention medium (%) and content as follows: starch: 3.0, K
2hPO
4: 0.12, l-asparagine: 0.11, extractum carnis: 0.05, wool: 0.2pH6.4.
Experimental result shows: the proteolytic enzyme that K20 bacterium produces to sheepskin, pigskin and ox-hide that process hides is conventional all have good depilatory active (
table 3): the hair removal effect wherein for ox-hide and sheepskin is best.Pelt after depilation is soft, has bating effect, and grain is level and smooth, and pore is clear.
table 3the protease depilation that bacterial strain produces is active
The evaluation of+hair removal effect ,+more, hair removal effect is better
The proteolytic enzyme adopting the bacterial strain for preparing of the present invention to produce to Sichuan Native Breed of Goats hair,
francemilk ox-hide and Australia oxhide carry out depilation process, and pelt after depilation is soft, has bating effect, and grain is level and smooth, pore clear (
as Fig. 7-
fig. 9shown in).
Claims (10)
1. a bacillus amyloliquefaciens, is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 14th, 2015, and preserving number is CGMCCNo.11067.
2. a preparation method for bacillus amyloliquefaciens, is characterized in that, comprises the following steps:
Step 1, sample collecting: sewer outlet place of tannery is with the earth of putrefying animal hair;
Step 2, enrichment: by sample access enrichment medium in cultivate 48h, proceed to liquid seed culture medium;
The primary dcreening operation of step 3, bacterial strain: after liquid seed culture medium cultivates 12h, by bacteria suspension streak culture 24h on casein Selective agar medium, picking has the even dibbling of single bacterium colony of hydrolysis circle in casein Selective agar medium, and 37 DEG C of constant temperature culture 48 hours, observe colony growth situation and casein is hydrolyzed situation;
The multiple sieve of step 4, bacterial strain: the even dibbling of the picking hydrolysis obvious bacterium colony of circle is on solid wool substratum, 37 DEG C of constant temperature culture more than 24 hours, observation colony growth situation and casein are hydrolyzed situation, then the single bacterium colony of picking hydrolysis circle more than 1 centimetre, 12 hours are activated in seed culture medium, inoculation by 6% measures bacterium liquid in fermention medium, 37 DEG C of shaker fermentations 36 hours, and fermented liquid is centrifugal;
Step 5,16sDNA sequence: carry out 16SrDNA sequence to the K20 bacterium filtered out, its sequence, as shown in SEQIDNO.1, prepares bacillus amyloliquefaciens.
3. the preparation method of bacillus amyloliquefaciens according to claim 2, is characterized in that, component and the content of described enrichment medium are as follows: peptone: 1.0%, extractum carnis: 1.0%, NaCl:0.5%, K
2hPO
4: 0.07%, casein: 1.0%.
4. the preparation method of bacillus amyloliquefaciens according to claim 2, is characterized in that, described primary dcreening operation substratum is liquid seed culture medium, its component and content as follows: glucose: 1.5%, K
2hPO
4: 0.07%, l-asparagine: 0.09%, extractum carnis: 0.07%.
5. the preparation method of bacillus amyloliquefaciens according to claim 2, is characterized in that, component and the content of described casein Selective agar medium are as follows: glucose: 1.5%, K
2hPO
4: 0.07%, l-asparagine: 0.09%, extractum carnis: 0.07%, casein food grade: 2.0%, agar: 5.0%.
6. the preparation method of bacillus amyloliquefaciens according to claim 2, is characterized in that, component and the content of described solid wool substratum are as follows: glucose 1.5%, extractum carnis 0.1%, l-asparagine 0.11%, K
2hPO
40.12%, wool 0.2%, agar: 5%, pH6.4.
7. the preparation method of bacillus amyloliquefaciens according to claim 2, is characterized in that, component and the content of described fermention medium are as follows: starch: 3.0%, K
2hPO
4: 0.12%, l-asparagine: 0.11%, extractum carnis: 0.05%, wool: 0.2%, pH6.4.
8. the proteolytic enzyme produced by bacillus amyloliquefaciens according to claim 1, is characterized in that, prepares by the following method: bacillus amyloliquefaciens fermention medium is through 72 hours fermentation, and what obtain contains protease fermented liquid.
9. the proteolytic enzyme of bacillus amyloliquefaciens generation according to claim 8, it is characterized in that, component and the content of described fermention medium are as follows: starch: 3.0%, K
2hPO
4: 0.12%, l-asparagine: 0.11%, extractum carnis: 0.05%, wool: 0.2%, pH6.4.
10. the application of the proteolytic enzyme produced by bacillus amyloliquefaciens according to claim 8 in process hides depilation.
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| 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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