CN105132397A - Method for improving soybean meal degrading efficiency - Google Patents
Method for improving soybean meal degrading efficiency Download PDFInfo
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- CN105132397A CN105132397A CN201510555686.5A CN201510555686A CN105132397A CN 105132397 A CN105132397 A CN 105132397A CN 201510555686 A CN201510555686 A CN 201510555686A CN 105132397 A CN105132397 A CN 105132397A
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- soybean meal
- enzyme
- sumizyme
- efficiency
- degrading
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- 238000000034 method Methods 0.000 title abstract description 11
- 235000019764 Soybean Meal Nutrition 0.000 title abstract description 7
- 239000004455 soybean meal Substances 0.000 title abstract description 7
- 230000000593 degrading effect Effects 0.000 title abstract 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 19
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 19
- 108091005804 Peptidases Proteins 0.000 claims description 9
- 239000013612 plasmid Substances 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
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- 239000002253 acid Substances 0.000 description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000004456 rapeseed meal Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Abstract
The invention aims at providing a method for improving soybean meal degrading efficiency so as to effectively improve soybean meal degrading efficiency. In order to improve the efficiency of alkaline protease in degrading soybean meal, the invention provides alkaline protease which is capable of efficiently degrading the soybean meal into polypeptide, wherein the amino acid sequence of the alkaline protease is as shown in SEQ ID NO: 4, and the sequence of a coding gene is as shown in SEQ ID NO: 3. The method disclosed by the invention, which prepares recombinant bacillus subtilis by virtue of the optimized alkaline protease, can be used for completely fermenting the soybean meal within a shorter time, so as to effectively improve fermenting efficiency and save cost.
Description
Technical field
The invention belongs to technical field of enzyme engineering, be specifically related to a kind of method improving beans Hectometer degradation efficiency.
Background technology
Dregs of beans is a kind of byproduct obtained after soybean extracting bean oil, is that in the animals and plants oil meal feeds products such as cottonseed meal, peanut meal, rapeseed meal, output is maximum, the one that purposes is the widest.As a kind of high protein, dregs of beans is the main raw material making livestock and poultry feed, and the dregs of beans of about 85% is used to the feed of poultry, aquatic animal.
But the not yet full grown cub of digestive ferment system (particularly for the animal of early weaning) can produce anaphylaxis to the antinutritional factor existed in beans Hectometer, more weak to the feed digestion ability using beans Hectometer as vegetable protein sources.And the existence of the nutritional autagonism factor is also affect the principal element that soybean protein source uses in feed.And in soybean peptides, be rich in many little peptides, can directly be absorbed by animal, and soybean peptides antigenicity is lower, after cub uses, anaphylactoid probability occurs and greatly reduce.Research shows to wean in early days in pigling feed and adds the substitute of soybean peptides as plasma protein powder, and it daily increases quality rate, feed coefficient, specific growth rate etc. without showing sex differernce.But make degradation of soybean meal be also there is the lower problem of hydrolysis efficiency in the process of the little peptide easily absorbed at present.
Summary of the invention
The object of this invention is to provide a kind of method improving beans Hectometer degradation efficiency, the enzymolysis efficiency of beans Hectometer is effectively provided.
In order to improve the degradation efficiency (using degree of hydrolysis as judgement criteria) of Sumizyme MP to beans Hectometer, the sequence of applicant to Sumizyme MP is transformed, obtain efficient degradation beans Hectometer to be the Sumizyme MP of polypeptide, the aminoacid sequence of this Sumizyme MP is SEQIDNO:4, and the sequence of its a kind of encoding gene is SEQIDNO:3.
The present invention also provides a kind of subtilis (Bacillussubtilis) of restructuring, is to prepare after the recombinant plasmid transformed carrying above-mentioned encoding gene;
On the other hand, applicant to be fermented beans Hectometer with the subtilis of this restructuring.
Sumizyme MP after the present invention uses optimization to prepare recombined bacillus subtilis, thus ferments completely to beans Hectometer in the time shorter again, thus effectively raises fermentation efficiency, provides cost savings.
Embodiment
Protein hydrolysis degree (Degreeofhydrolysis, DH) of the present invention refers to the percentage ratio that the peptide bond in protein is hydrolyzed, and its calculation formula is as follows:
DH％＝h/h
totx100％
H be peptide bond cleaved in every gram of albumen after proteolysis mmole number (mmlo/g protein),
H
tot, take casein as standard, its value is 8.2mmlo/g protein;
When the peptide bond of protein is all hydrolyzed the amino acid generating and dissociate, then degree of hydrolysis is 100%; When not being hydrolyzed, its degree of hydrolysis is 0.Due to cracking every during proteolysis peptide bond just newly-generated-NH
2with-COOH a base, therefore, when measuring the degree of hydrolysis of protein, if quantitative mensuration newly-generated-NH
2just h value can be calculated with-COOH base unit weight; The degree of hydrolysis that so just can calculate protein comes.
The present invention adopts formaldehyde fixation to measure h value (National Standard of the People's Republic of China's formolite number method GB12143.2-89).
Below in conjunction with example, method of the present invention is described further, the experimental technique of unreceipted actual conditions in embodiment, usually can condition routinely, condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write, or run according to the condition that manufacturer advises.
The optimization of embodiment 1 Sumizyme MP
1, utilize the technology of fallibility PCR to be classified as to nucleotides sequence in vitro in the alkaline protease gene (the mature peptide aminoacid sequence of coding is SEQIDNO:2) of SEQIDNO:1 and introduce coding mutation, the system of fallibility PCR is as follows:
10 × PCRBuffer is not (containing MgCI
2) 5 μ L, dCTP (25mmol/L) 2 μ L, dTTP (25mmol/L) 2 μ L, dGTP (10mmol/L) 1 μ L, dATP (10mmol/L) 1 μ L, F (10pmol/ μ L) 1 μ L, R (10pmol/ μ L) 1 μ L, Mg
2+(20mM) 14 μ L, Mn
2+(3mM) 1.5 μ L, pQC670.5 μ L, TaqDNApolymerase (2.5U) 1 μ L, ddH
2o20 μ L.
Upstream primer: 5 '-tagttcatcgatcgcatcggctgctgaagaagcaaaagaaaaat-3 '
Downstream primer: 5 '-atttttcttttgcttcttcagcagccgatgcgatcgatgaacta-3 ';
Amplification condition is: 94 DEG C of 10min; 94 DEG C of 60s, 58 DEG C of 60s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 10min.GelExtractionKit is utilized to reclaim mutant pcr amplification product.
2, the variants obtained that increases is connected with expression vector
With pQC67 plasmid for template, utilize
Upstream primer: tgcagaagcggcaacacgctaaattaagcatgcaagctagttgc
Carry out pcr amplification with downstream primer atttttcttttgcttcttcagcagccgatgcgatcgatgaacta as primer, obtain vector gene sequence.Amplification condition is: 98 DEG C of 10min; 98 DEG C of 10s, 55 DEG C of 20s, 72 DEG C of 3min, 30 circulations; 72 DEG C of 10min.GelExtractionKit is utilized to reclaim pcr amplification product.
The mutant pcr amplification product of recovery and carrier segments are formed polymer by Overlap extension PCR, amplification system is as follows: 5 × PhusionHFBuffer10 μ L, 2.5mMdNTPs8 μ L, Insertgene (aprE fragment) 4 μ L, Linearizedvector (pQC67) 6 μ L, PhusionDNAPolymerase1 μ L, ddH
2o21 μ L.Amplification condition is 98 DEG C of 10min; 98 DEG C of 10s, 72 DEG C of 3min, 20 circulations; 98 DEG C of 10s, 72 DEG C of 6min, 15 circulations; 72 DEG C of 10min.
Polymer is transformed Bacillussubtilis1A751 Host Strains, obtain positive transformant, be the recombinant bacterial strain (experimental strain) expressing alkali protease mutation body.It is simultaneously bacterial strain in contrast after the alkaline protease gene (wild-type) of SEQIDNO:2 builds with sequence.
3, the screening of alkali protease mutation body
Under aseptic condition, the recombinant bacterial strain obtained in step 2 is inoculated in respectively and adds in 50mL substratum test tube (seed culture medium consist of 0.5% yeast powder, 1% Tryptones, 1% glucose, 1.8%K
2hPO
4, maltodextrin 5 ~ 10%, Trisodium Citrate 0.1 ~ 0.5%, CaCl
20.1 ~ 0.5%, MgSO
40.1 ~ 0.5%, K
2hPO
40.5 ~ 2%) in, 34 DEG C, 210rpm shaking culture 8h; And then add the soybean protein isolate easy (SPI solution) that 30ml concentration is 12%, in 34 DEG C, 250rpm shaking culture 36h; Then carry out centrifugal, the supernatant liquor of acquisition measures hydrolysis angle value DH, thus filters out the recombinant bacterial strain strengthened soybean protein powder degradation capability.Finishing screen selects 2 recombinant bacterial strains obviously strengthened soybean protein powder degradation capability (compared to contrast strain,), called after Bacillus – a and Bacillus-b, wherein the DH value of recombinant bacterial strain Bacillus-a is 12, the DH value of recombinant bacterial strain Bacillus – b is 17, and the DH value of control strain is 8.But the degree of hydrolysis of the recombinant bacterial strain of the overwhelming majority and control strain does not have difference, and the DH value of some recombinant bacterial strains reduces than control strain; The result repeating to test is also identical.
The determination of the bacterial strain neutral and alkali Protease sequences that embodiment 2 filters out
Plasmid extraction kit is utilized to extract plasmid in Bacillus – a and Bacillus – b bacterial strain respectively; By Shanghai, Sheng Gong company carries out gene sequencing respectively to above-mentioned plasmid.Sequencing result shows: the nucleotides sequence of the alkaline protease gene that the plasmid in Bacillus – a bacterial strain carries is classified as SEQIDNO:3, and the aminoacid sequence of its coding is SEQIDNO:4, called after Ba-enzyme; The nucleotides sequence of the alkaline protease gene that the plasmid in subtilis Bacillus – b carries is classified as SEQIDNO:5, and the aminoacid sequence of its coding is SEQIDNO:6, called after Bb-enzyme.Gene comparision is found, the aminoacid sequence (SEQIDNO:4) of the Sumizyme MP Ba-enzyme that Bacillus – a obtains is compared with the Sumizyme MP SEQIDNO:2 of wild-type, 41st amino acids sports Ser by Leu, and the 101st amino acids sports Leu by Ser; 201st amino acids sports ASN by Ser.And the aminoacid sequence of Bb-enzyme (SEQIDNO:6) is compared with the Sumizyme MP SEQIDNO:2 of wild-type, 65th amino acids sports Leu by His, 125th amino acids sports Val by Gly, and the 148th amino acids sports Leu by Val; 205th amino acids sports Val by Gly.
Simultaneously, applicant does not have difference with the degree of hydrolysis of control strain yet, and analyze than the sequence of the Sumizyme MP in the recombinant bacterial strain of control strain reduction, found that, wherein also there is one or more amino acid whose sudden change in the Sumizyme MP of some bacterial strain compared with original basic protein enzyme sequence.
The recombinant expressed rear characterization analysis of embodiment 3:Ba-enzyme, Bb-enzyme enzyme
To carry energy Ba-enzyme, Bb-enzyme bacterial strain, and control strain carries out abduction delivering, obtains Ba-enzyme and Bb-enzyme after purifying, and the recombinant basic proteolytic enzyme of control group, degree of being hydrolyzed detects as follows:
Compound concentration is that each 200mL of SPI solution of 4% (w/v) is in 50 DEG C of pre-treatment 10min, 50 DEG C, each 5g of original alkaline proteolytic enzyme that adds Ba-enzyme, Bb-enzyme and contrast under pH10.0 condition carries out enzymolysis, the mensuration of enzymolysis 40min degree of being hydrolyzed, 3 Duplicate Samples are set, average.Result shows, the DH value of the original alkaline proteolytic enzyme of contrast is the DH value of 14, Ba-enzyme is 22, and the DH value of Bb-enzyme is 26, shows that the enzyme suddenlyd change has better effect than protoenzyme.
The enzyme of Ba-enzyme, Bb-enzyme is lived simultaneously and measure, adopt National Standard of the People's Republic of China's protease preparation measuring method (GB/T25327-2009).Folin's methods is used to measure the vigor of proteolytic enzyme, the solution used comprises: forint uses solution, and (a commercially available folin solution mixes with two parts of water, shake up), sodium carbonate solution (42.4g/L), trichoroacetic acid(TCA) (65.4g/L), gradient pH value damping fluid, casein solution (10.0g/L).Reaction process is as follows: add 1mL enzyme liquid in test tube, and 40 DEG C of temperature bath 2min, add casein solution 1mL, shakes up rear 40 DEG C of temperature bath 10min, adds 2mL solution of trichloroacetic acid, shake up (blank first adds trichoroacetic acid(TCA), then adds casein solution).Take out static 10min, qualitative filter paper filters at a slow speed.Get 1mL filtrate, add sodium carbonate solution 5mL, add Folin reagent and use solution 1mL, 40 DEG C of colour developing 20min, in 680nm wavelength, measure absorbancy with 10mm cuvette.Result shows that the enzyme work of Bb-enzyme is higher than Ba-enzyme, consistent with the result of degree of hydrolysis.
Ferment Ba-enzyme and Bb-enzyme bacterial strain beans Hectometer, bean cake powder is broken to below 40 orders, and dregs of beans and water mix in the ratio of 1:1.5, then inoculating strain in the mixture, at the condition bottom fermentation 36 hours of 35 DEG C, ferment with positive control strain inoculation simultaneously.Crude protein, trichoroacetic acid(TCA) soluble nitrogen (TCA-NSI) content, trypsin ihhibitor and urease activity is detected after fermentation.Result shows, compared to Positive contrast bacteria, Ba-enzyme and Bb-enzyme bacterial strain can make fermentation beans Hectometer reach service requirements as fodder additives within the shorter time, thus effectively provides cost savings, and fermentation after product is for the preparation of feed ingredient.
Claims (5)
1. a Sumizyme MP, is characterized in that, described Sumizyme MP includes:
1) aminoacid sequence is the Sumizyme MP of SEQIDNO:4;
2) with 1) Sumizyme MP there is 95% or more homology, and there is the proteolytic enzyme of alkaline protease activity.
2. a gene, is characterized in that, described genes encoding Sumizyme MP according to claim 1.
3. gene as claimed in claim 2, it is characterized in that, the sequence of described gene is SEQIDNO:3.
4. a recombined bacillus subtilis (Bacillussubtilis) is prepared after the recombinant plasmid transformed carrying gene according to claim 2.
5. a fermentation process of beans Hectometer, is characterized in that, described fermentation process recombined bacillus subtilis according to claim 4 ferments.
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| CN201810439755.XA CN108570462A (en) | 2015-09-05 | 2015-09-05 | A method of improving beans Hectometer degradation efficiencies |
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| CN105132397A true CN105132397A (en) | 2015-12-09 |
| CN105132397B CN105132397B (en) | 2018-07-06 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113729164A (en) * | 2021-06-08 | 2021-12-03 | 中新国际联合研究院 | Combined preparation method of acidic soybean polypeptide beverage and probiotic preparation |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN102676482A (en) * | 2012-05-11 | 2012-09-19 | 河南省南街村（集团）有限公司 | Method for producing alkali protease from highland bacillus |
| CN103829031A (en) * | 2013-07-05 | 2014-06-04 | 河南工业大学 | Soybean meal fermentation process |
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| CN105176952B (en) * | 2015-09-05 | 2018-05-18 | 云南神农农业产业集团股份有限公司 | A kind of alkali protease for improving beans Hectometer degradation efficiencies |
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| CN102676482A (en) * | 2012-05-11 | 2012-09-19 | 河南省南街村（集团）有限公司 | Method for producing alkali protease from highland bacillus |
| CN103829031A (en) * | 2013-07-05 | 2014-06-04 | 河南工业大学 | Soybean meal fermentation process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113729164A (en) * | 2021-06-08 | 2021-12-03 | 中新国际联合研究院 | Combined preparation method of acidic soybean polypeptide beverage and probiotic preparation |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN108570462A (en) | 2018-09-25 |
| CN105132397B (en) | 2018-07-06 |
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