CN103013960A - Alkaline protease and recombinant expression engineering bacterium thereof - Google Patents
Alkaline protease and recombinant expression engineering bacterium thereof Download PDFInfo
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- CN103013960A CN103013960A CN2012105635401A CN201210563540A CN103013960A CN 103013960 A CN103013960 A CN 103013960A CN 2012105635401 A CN2012105635401 A CN 2012105635401A CN 201210563540 A CN201210563540 A CN 201210563540A CN 103013960 A CN103013960 A CN 103013960A
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Abstract
The invention relates to the technical field of microorganism engineering, in particular to alkaline protease and a recombinant expression engineering bacterium thereof. An alkaline protease gene of bacillus clausii is transferred into bacillus subtilis to construct a bacillus subtilis engineering strain with the preservation number of CCTCC (China Center for Type Culture Collection) No: M2012499. The engineering strain can express the alkaline protease efficiently, and the enzymatic activity of the engineering strain fermented in a 20L tank is as high as 9120U/ml. The recombinant expression alkaline protease can widely serve as an additive for a washing article.
Description
Technical field
The invention belongs to the microbial engineering field, be specifically related to a kind of Sumizyme MP and recombinant expressed engineering bacteria thereof.
Background technology
Sumizyme MP (Alkaline protease) refers to the enzyme of protein hydrolysate peptide bond in pH value meta-alkalescence scope.Sumizyme MP is how to be got by fermentation of bacillus, and main component is a kind of restriction endonuclease proteolytic enzyme, and catalytic site is Serine, and molecular weight is about 27kDa.Domestic industry is produced protease strain and is mostly through subtilis 2709 seed selections.By submerged fermentation, extraction and the refining a kind of proteolytic ferment that forms, its can generate polypeptide or amino acid by protein hydrolysate molecule peptide chain, has the ability of stronger decomposing protein.
Industry (Zheng Huanyu etc., 2003, the soybean circulars such as Sumizyme MP is widely used in food, medical treatment, brewages, silk, process hides; Xia Liangshu etc., 1998, institute of Zhongnan Polytechnic College newspaper).Sumizyme MP is the enzyme that occupies the ratio maximum in the industrial enzymes, accounts for greatly about 60% (Mala B. R. etc., 1998, Microbiology and Molecular Biology Reviews) of the annual total sales volume in the whole world; Be popular in the market detergent additive, can increase substantially the washing soil removability, especially to protein dirts such as bloodstain, sweat stain, milk stain, oil stains, have unique washing effect.
The research of domestic basic protein bacterium focuses mostly in the seed selection of superior strain and evaluation (Huang Jixiang, 2011, microbiology circular; Yellow will is strong etc., 2006, Fujian Agricultural Univeristy's journal), and fermentation condition optimization etc. (Tang Xueming etc., 2002, biotechnology journal).At present, how domestic industry produces bacterium by Bacillus subtilus 2709 seed selections; These shortcomings of producing bacterium are: fermenting enzyme live lower (＜35000U/ml); In aftertreatment, it is more and with special bad smell that protein product often contains impurity behind the salt precipitation.Simultaneously, Liang Xiaomei etc. (2011, the biotechnology circular) attempt to clone highly active protein enzyme gene by having made up high flux screening.The directive breeding of Research for Industrial Microbial Germ is mainly carried out in the seed selection of external Sumizyme MP superior strain with genetic engineering technique and protein engineering means, purpose is strong, and enzymatic structure is studied to get deeply (Tsuyoshi N etc., 2004, the J. Biol. Chem.) that also compare.
Sumizyme MP on the current washing market by Novi letter and outstanding can the monopolization of two large zymin international corporations of section, its product enzyme is lived high, dissolution rate during soon and with the washing composition compatibility tolerance strong.Domestic zymin enterprise will enlarge the market share in the detersive enzyme field, must accelerate development go out the fermenting enzyme novel alkali proteinase high, that production cost is low of living, and improves the competitive power of self.
Summary of the invention
The purpose of this invention is to provide a kind of Sumizyme MP and recombinant expressed engineering strain thereof.The present invention changes in the subtilis (Bacillus subtilis) by the alkaline protease gene with Bacillus clausii (Bacillus clausii), makes up to obtain the subtilis engineering strain.Described engineering strain energy efficient secretory expression Sumizyme MP is for low-cost, large-scale production Sumizyme MP are laid a good foundation.
One aspect of the present invention relates to a kind of Sumizyme MP that separates from Bacillus clausii, described Sumizyme MP includes:
1) aminoacid sequence is the Sumizyme MP of SEQ ID NO:1;
2) in the amino acid that in SEQ ID NO:1, limits through replacing, lack or add one or several amino acid and have 1) the neutral and alkali protease function, the protein of being derived by SEQ ID NO:1.
The encoding gene of the Sumizyme MP of above-mentioned Bacillus clausii, its nucleotides sequence are classified SEQ ID NO:2 as.
The invention still further relates to be used to the expression vector of expressing above-mentioned Sumizyme MP, described expression vector carries the encoding gene that sequence is SEQ ID NO:2.
Another aspect of the present invention provides a kind of subtilis engineering bacteria, and this project bacterium carries the expression vector of the Sumizyme MP of expressing Bacillus clausii (Bacillus clausii); This subtilis AP1(Bacillus subtilis AP1), be preserved in the Chinese Typical Representative culture collection center that is positioned at Lopa Nationality an ancient woman's ornament mountain, Wuhan Wuhan University on December 5th, 2012, preserving number is CCTCC NO:M2012499.
The present invention relates to the application of above-mentioned subtilis engineering bacteria on the other hand, for the production of Sumizyme MP.
Subtilis engineering strain of the present invention can efficiently express Sumizyme MP, and enzyme work reaches 9120U/mL.The optimal pH of described Sumizyme MP is 12.0, and optimum temperuture is 60 ℃, can be widely used as the articles for washing additive.
Description of drawings
Fig. 1: the genetic map of the carrier that the present invention is used.
Fig. 2: the SDS-PAGE of shake flask fermentation supernatant detects electrophorogram, and arrow indication place is recombinant expressed Sumizyme MP.
Fig. 3: the product enzyme graphic representation of 20L tank fermentation.
Embodiment
Below in conjunction with example method of the present invention is described further, the experimental technique of unreceipted actual conditions among the embodiment, usually condition routinely, condition described in " the molecular cloning experiment guide " write such as J. Pehanorm Brooker (Sambrook) etc., or the condition operation of advising according to manufacturer.The relevant technician in this area can understand better and grasps the present invention by embodiment.But, the case that protection of the present invention and claim scope are not limited to provide.
The clone of embodiment 1 Bacillus clausii alkaline protease gene
1.1 the extraction of genomic dna
The TIANamp Bacteria DNA Kit of TIAN GEN company prepares the genomic dna of Bacillus clausii, the operational manual of its preparation process reference reagent box.
1.2 gene clone
The genome DNA of extracting in 1.1 utilizes primer 1:5 '-TGGAGAAAGGGCGAAGACGAA-3 ' and primer 2: 5 '-CTACCATTTGATGAACGGAGC-3 ' to carry out pcr amplification as masterplate.The pcr amplification condition is 98 ℃ of 10min; 98 ℃ of 10s, 68 ℃ of 4min, 30 circulations; 72 ℃ of 10min.Utilize E. Z. N. A. Gel Extraction Kit to reclaim pcr amplification product.
1.3 sequencing analysis
The amplified production that reclaims in 1.2 is connected to TAKARA pMD18-T carrier, corresponding cloning vector called after pT-aprE; At last positive colony is delivered to the Beijing Liuhe Huada Genomics Technology Co., Ltd and carry out sequencing analysis.Sequencing result shows that pcr amplification is errorless, and the nucleotides sequence of amplified production is classified SEQ ID NO:3 as, and sequence is through BLAST compare of analysis this genes encoding Sumizyme MP as can be known.
The structure of embodiment 2 Sumizyme MP expression vectors and engineering bacteria
2.1 the structure of expression vector
Nucleotide sequence according to shown in the SEQ ID NO:3, design following primer:
Primer 3:5 '-ACGCGTCGACGATGAAGAAACCGTTGGGGAAA-3 ' (SalI restriction enzyme site)
Primer 4:5 '-ATTAGCAACGTGCATACCATTGTTCCCTGCC-3 '
Primer 5:5 '-GGCAGGGAACAATGGTATGCACGTTGCTAAT-3 '
Primer 6:5 '-ACATGCATGCGCGGTCTGTTCTCTTTTTCGA-3 ' (SphI restriction enzyme site)
Take the genomic dna of Bacillus clausii as masterplate, utilize primer 3, primer 4 pcr amplification Sumizyme MP structure gene initiator codons to the sequence between the inner SphI restriction enzyme site, obtain fragment aprE-U.The pcr amplification condition is 98 ℃ of 10 min; 98 ℃ of 30 s, 68 ℃ of 1 min, 30 circulations; 72 ℃ of 10 min.Utilize primer 5, the inner SphI restriction enzyme site of primer 6 pcr amplification Sumizyme MP structure genes to the sequence between the terminator, obtain fragment aprE-D.The pcr amplification condition is 98 ℃ of 10 min; 98 ℃ of 10 s, 68 ℃ of 1 min, 30 circulations; 72 ℃ of 10 min.Utilize E. Z. N. A. Gel Extraction Kit to reclaim aprE-U, aprE-D fragment.
AprE-U, aprE-D fragment are obtained to merge fragment aprE by merging PCR.It is as follows to merge the PCR process: the first round, respectively add aprE-U, the aprE-D fragment of 200 ng in the PCR reaction system, and do not add primer, 10 circulations of pcr amplification, the pcr amplification condition is 98 ℃ of 10 min; 98 ℃ of 10 s, 68 ℃ of 3 min, 10 circulations; 72 ℃ of 10 min.Second takes turns, and gets 10 μ l first round PCR products as template, utilizes primer 3,30 circulations of primer 6 pcr amplifications, and the pcr amplification condition is 98 ℃ of 10 min; 98 ℃ of 10 s, 68 ℃ of 2 min, 30 circulations; 72 ℃ of 10 min.Utilize E. Z. N. A. Gel Extraction Kit to reclaim the PCR product of about 1.1 kb, obtain the aprE fragment.Through order-checking, the nucleotides sequence of this aprE fragment is classified SEQ ID NO:2 as, and its encoding amino acid sequence is SEQ ID NO:1.A plurality of sequencing results are all consistent, prove the mistake that does not occur in the amplification.
With aprE fragment SalI, SphI double digestion, simultaneously the pWB980 carrier is also carried out SalI, SphI double digestion, after utilizing E. Z. N. A. Gel Extraction Kit purifying, with T4 DNA Ligase aprE/SalI/SphI is connected with pWB980/SalI/SphI, to connect product and transform Bacillus subtilis 1A751 Host Strains, obtain positive transformant, after the sequence verification, with the plasmid called after pWB980-protease that obtains, plasmid map as shown in Figure 1.
Bacillus subtilis 1A751 utilizes the competence method to transform, the conversion detailed process is as follows: with the Bacillus subtilis 1A751 of fresh activation by LB(Tryptones 1%, yeast powder 0.5%, NaCl 1%) (GM I compound method is plating: the minimum salts solution 95.6ml of 1* to 5 ml GM I, 20% glucose, 2.5 ml, 5% caseinhydrolysate, 0.4 ml, 10% yeast powder juice 1ml; Wherein the compound method of the minimum salts solution of 1* is: K
2HPO
414 g/L, KH
2PO
46 g/L, (NH
4)
2SO
42 g/L, trisodium citrate 1 g/L, MgSO
47H
2O 0.2 g/L, successively dissolving in distilled water) solution, spend the night at 30 ℃, 125 rpm shaking culture.Second day is got 2 ml and is transferred in the 18 ml GM I, and 37 ℃, 250 rpm are cultivated 3.5 h.The nutrient solution of getting again 10 ml previous steps is transferred to 90 ml GM II, and (GM II compound method is: minimum salts solution 96.98 ml of 1*, 20% glucose, 2.5 ml, 5% caseinhydrolysate, 0.08 ml, 10% yeast powder juice, 0.04 ml, 1 M MgCl
20.25 ml, 1 M CaCl
20.05 ml), after 37 ℃, 125 rpm are cultivated 90 min, the centrifugal collection thalline of 5000 g, 10 min.With the 10 ml original fluid supernatant liquors thalline that suspends gently, the thalline after the suspension is competent cell.Then in 0.5 ml competence, add an amount of DNA and behind 37 ℃, 200 rpm shaking culture, 30 min, be coated with corresponding resistant panel (LB), again 37 ℃ of overnight incubation, inferior daily inspection and checking transformant.
PWB980-protease transforms the engineering bacteria called after subtilis AP1(Bacillus subtilis AP1 that obtains behind the Bacillus subtilis 1A751), and be preserved on December 5th, 2012 that " Chinese Typical Representative culture collection " center ", deposit number are CCTCC NO:M 2012499.Fermentation results shows that the recombinant bacterial strain of preservation can express Sumizyme MP efficiently, and the work of 32 hours Sumizyme MP enzymes of fermentation is about 9120U/ml.And, also can guarantee the expression level of Sumizyme MP after repeatedly going down to posterity.
Embodiment 3 fermentations and zymologic property are measured
3.1 enzyme activity determination method
Use folin's methods to measure the albumen enzyme activity, the solution that uses comprises: forint is used solution (a commercially available folin solution mixes with two parts of water, shakes up), sodium carbonate solution (42.4 g/L), trichoroacetic acid(TCA) (65.4 g/L), gradient pH value damping fluid, casein solution (10.0 g/L).Reaction process is as follows: add 1ml enzyme liquid in the test tube, 40 ℃ of temperature are bathed 2min, add casein solution 1ml, shake up rear 40 ℃ of temperature and bathe 10min, add 2ml trichoroacetic acid(TCA) solution, shake up (blank adds first trichoroacetic acid(TCA), adds casein solution again).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 ℃ of colour developing 20min in the 680nm wavelength, measure absorbancy with the 10mm cuvette.
The protease activity unit of force is defined as 1g solid enzyme powder (or 1 ml liquid enzymes), and under certain temperature and pH value condition, the 1min caseinhydrolysate produces 1 μ g tyrosine, is 1 enzyme activity unit.
3.2 shake flask fermentation
(yeast soaks powder 0.5%, Tryptones 0.5%, glucose 1%, K in the 50ml seed culture medium from the single colony inoculation of the dull and stereotyped picking Bacillus of the LB that contains 25 μ g/ml kantlex subtilis AP1
2HPO
41.8%, kantlex 25 μ g/ml) in, 34 ℃, 210 rpm shaking culture 8h.Get 2.5ml be inoculated into the 50ml fermention medium (Tryptones 1%, yeast powder 0.5%, NaCl1%) in, 34 ℃, 250rpm shaking culture 48h, get supernatant liquor and carry out the SDS-PAGE electrophoresis detection, the result as shown in Figure 2, arrow indication place is recombinant expressed Sumizyme MP.
3.3 20L tank fermentation
(yeast soaks powder 0.5%, Tryptones 0.5%, glucose 1%, K in 0.6 L seed culture medium from the single colony inoculation of the dull and stereotyped picking Bacillus of the LB that contains 25 μ g/ml kantlex subtilis AP1
2HPO
41.8%, kantlex 25 μ g/ml) in, 34 ℃, 210rpm shaking culture 8h.The 0.6L seed culture fluid is inoculated in the 20L fermentor tank that contains 12L fermention medium (NaCl 1% for Tryptones 1%, yeast powder 0.5%) fully 34 ℃ of fermentation culture 44h fermentation ends; Wherein in the fermenting process, the sampling and measuring enzyme was lived in per 4 hours.Produce the enzyme curve as shown in Figure 3, the 32 hours Sumizyme MP enzymes that ferment are alive the highest, are about 9120U/ml.
3.2 zymologic property analysis
(1) optimal pH analysis
Be respectively 7.0,8.0,9.0,10.0,11.0,12.0,13.0 damping fluid with the pH value, measuring enzyme under 40 ℃ of conditions of temperature lives, take the highest enzyme work as 100%, calculating relative enzyme lives, do the relative enzyme of pH-curve alive, the result shows that the optimal pH of the Sumizyme MP that the present invention is recombinant expressed is 12.0.
(2) optimum temperuture analysis
At 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, measure enzyme under the condition of pH 10.5 and live respectively, take the highest enzyme work as 100%, calculate relative enzyme and live, do temperature-relative enzyme curve alive.The result shows that the optimum temperuture of the Sumizyme MP that the present invention is recombinant expressed is 60 ℃.
The application of embodiment 4 Sumizyme MPs in washing composition
Get above-mentioned Sumizyme MP fermented liquid and be concentrated into about 100000U/ml, add a certain amount of chemical stabilizer, sanitas etc., after dissolving mixes, be mixed with liquid protein zymin (concrete component sees Table 1).Measure enzyme activity after the liquid protein zymin is deposited certain hour (enzyme live conservation rate) with standard method (casein Folin reagent method), and order observes its sedimentation situation of survey, carry out the evaluation of stability.Measure the washing decontamination value of liquid enzyme formulation in liquid washing agent with national standard method simultaneously, relatively its actual clean effect.
Table 1 liquid basified protease component and content
| Sequence number | | Weight part | |
| 1 | The Sumizyme MP fermented liquid | 25?–?60 | |
| 2 | |
0?–?0.3 | |
| 3 | The boric acid or derivatives thereof | 0?–?3 | |
| 4 | |
0?–?10 | |
| 5 | Polyvalent alcohol | 5?–?30 | |
| 6 | Potassium sorbate | 0.1?–?0.5 |
With said components, under 40 ℃ of temperature, stirred 15-30 minute, fully dissolving after mixing, is cooled to room temperature, can make preferably liquid basified protease preparation of a kind of stability.
The liquid enzyme formulation that makes after depositing 30 days under 40 ℃, is observed and found that this enzymes soln produces without sedimentation.Through the enzyme activity test analysis, its enzyme activity conservation rate is still in (testing standard: light industry rower QB/T 1803-93 " industrial enzymes universal test method ") more than 95%.
The decontamination value of carrying out under equal enzyme (1000u/ml) dosage condition alive with this liquid basified protease preparation and market competition product simultaneously, compares (testing standard: GB GB/T 13174-2008 " the dress material mensuration of washing composition detersive power and circulation cleaning performance ").
The decontamination pH-value determination pH data of table 2 different liqs Sumizyme MP in commercially available washing liquid
| Numbering | The sample title | EMP?A116 | EMP? |
| 1 | Blue moon washing liquid 2g+1000u/ml Sumizyme MP of the present invention | 8.00? | 8.53? |
| 2 | Blue moon washing liquid 2g+1000u/ml competing product Sumizyme MP | 6.91? | 7.74? |
| 3 | Blue moon washing liquid 2g+2000u/ml competing product Sumizyme MP | 7.63? | 8.65? |
| 4 | National standard washing liquid 2g(does not add proteolytic enzyme) | 2.13? | 1.71? |
Can be found out by the data in the table: under the equal enzyme dosage alive, liquid basified protease of the present invention is better than the competing product Sumizyme MP to the clean effect on international soiled cotton EMPA116 and the EMPA117.
The recombinant expressed Sumizyme MP of engineering bacteria of the present invention can increase substantially the washing soil removability as detergent additive, especially to protein dirts such as bloodstain, sweat stain, milk stain, oil stains, has unique washing effect.
Claims (5)
1. Sumizyme MP, described Sumizyme MP includes:
1) aminoacid sequence is the albumen of SEQ ID NO:1;
2) in the amino acid that in SEQ ID NO:1, limits through replacing, lack or add one or several amino acid and have 1) the neutral and alkali protease function, the protein of being derived by SEQ ID NO:1.
2. for the encoding gene of the Sumizyme MP claimed in claim 1 of encoding, its nucleotides sequence is classified SEQ ID NO:2 as.
3. be used for expressing the recombinant expression vector of Sumizyme MP claimed in claim 1; Described recombinant expression vector carries the encoding gene that sequence is SEQ ID NO:2.
4. subtilis engineering bacteria that is used for expressing Sumizyme MP claimed in claim 1, its deposit number is CCTCC NO:M2012499.
5. the application of subtilis engineering bacteria claimed in claim 4 in producing Sumizyme MP.
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Cited By (7)
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| CN103789227B (en) * | 2013-12-04 | 2015-04-15 | 青岛蔚蓝生物集团有限公司 | High-alkaline protease yield bacillus subtilis strain |
| CN105018402A (en) * | 2015-07-13 | 2015-11-04 | 青岛蔚蓝生物集团有限公司 | Bacillus licheniformis capable of producing alkaline protease in large quantity and application of Bacillus licheniformis |
| CN108085327A (en) * | 2017-12-28 | 2018-05-29 | 山东大学 | A kind of alkali protease heterogenous expression engineered strain in extreme environment source and its application |
| CN108441489A (en) * | 2018-04-09 | 2018-08-24 | 青岛蔚蓝生物集团有限公司 | A kind of bacillus subtilis of protein production processes and high yield alkali protein |
| CN110904002A (en) * | 2019-11-25 | 2020-03-24 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN111893126A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Alkaline protease gene, alkaline protease, preparation method and application thereof |
| 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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| CN103789227B (en) * | 2013-12-04 | 2015-04-15 | 青岛蔚蓝生物集团有限公司 | High-alkaline protease yield bacillus subtilis strain |
| CN105018402A (en) * | 2015-07-13 | 2015-11-04 | 青岛蔚蓝生物集团有限公司 | Bacillus licheniformis capable of producing alkaline protease in large quantity and application of Bacillus licheniformis |
| CN108085327A (en) * | 2017-12-28 | 2018-05-29 | 山东大学 | A kind of alkali protease heterogenous expression engineered strain in extreme environment source and its application |
| CN108085327B (en) * | 2017-12-28 | 2021-02-19 | 山东大学 | Alkaline protease heterologous expression engineering strain from extreme environment and application thereof |
| CN108441489A (en) * | 2018-04-09 | 2018-08-24 | 青岛蔚蓝生物集团有限公司 | A kind of bacillus subtilis of protein production processes and high yield alkali protein |
| CN108441489B (en) * | 2018-04-09 | 2023-04-14 | 潍坊康地恩生物科技有限公司 | Protein production method and bacillus subtilis for high-yield production of alkaline protease |
| CN110904002A (en) * | 2019-11-25 | 2020-03-24 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN110904002B (en) * | 2019-11-25 | 2022-04-29 | 天津大学 | Method for biologically removing tetracycline antibiotics |
| CN111893126A (en) * | 2020-07-01 | 2020-11-06 | 深圳润康生态环境股份有限公司 | Alkaline protease gene, alkaline protease, preparation method and application thereof |
| 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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