CN102827816A - Alpha-amylase and application thereof - Google Patents
Alpha-amylase and application thereof Download PDFInfo
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- CN102827816A CN102827816A CN2012102815328A CN201210281532A CN102827816A CN 102827816 A CN102827816 A CN 102827816A CN 2012102815328 A CN2012102815328 A CN 2012102815328A CN 201210281532 A CN201210281532 A CN 201210281532A CN 102827816 A CN102827816 A CN 102827816A
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- 108090000637 alpha-Amylases Proteins 0.000 title abstract description 17
- 102000004139 alpha-Amylases Human genes 0.000 title abstract description 15
- 229940024171 alpha-amylase Drugs 0.000 title abstract description 15
- 235000008429 bread Nutrition 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 11
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
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- 238000003259 recombinant expression Methods 0.000 claims description 5
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- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010023063 Bacto-peptone Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001147665 Foraminifera Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 241000408008 Talaromyces marneffei ATCC 18224 Species 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
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- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 108010081495 driselase Proteins 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to alpha-amylase and application thereof. The nucleotide sequence of the alpha-amylase is SEQ ID No. 2, and the amino acid sequence is SEQ ID No. 1. The alpha-amylase screened in the invention plays a critical role in hydrolysis of starch into maltose, low oligosaccharides and a small amount of glucose, can be used for production of high maltose syrup and for production of baked products like bread as an additive, is applied in the industries like beers, yellow rice wines and raw alcohol to some extent and has an industrial production application value. Furthermore, the screened alpha-amylase can be used for transforming engineering bacteria so as to obtain recombinant bacteria expressing the alpha-amylase.
Description
Technical field
The invention belongs to the enzyme gene engineering technology field, be specifically related to a kind of AMS and application thereof.
Background technology
The AMS of originated from fungus can be rough be divided into 3 types by zymologic property or action condition: neutral fungal alpha-amylase, acidproof or thermotolerance fungal alpha-amylase, have the fungal alpha-amylase of living amylase activity.AMS is an important industrial enzymes, and it is used to produce product such as high maltose syrup, baked goods.In industries such as beer brewing industry (improving the fermentability of wort), yellow rice wine brew industry (improve vinosity, improve the yield of liquor) and oligomeric isomaltose productions application is in various degree arranged all.
Reported that multiple fungal alpha-amylase gene obtains expressing in different hosts; Wherein the heterogenous expression of fungal alpha-amylase gene mainly concentrates on eukaryotic expression system; Although the expression level of heterogenous expression fungal alpha-amylase is not high in most reports, heterogenous expression is the preferred approach that realizes that the fungal alpha-amylase single enzyme is lived.
Though China is carrying out laterly aspect the research of fungal alpha-amylase, achievement in research has positive effect to promoting China's zymin industry.Compare with the research level of other amylolytic enzyme system; The research level of China's fungal alpha-amylase is not high; All researchs almost do not relate to the cloning and expression of gene; The fermentative prodn mode does not change to liquid fermenting from solid state fermentation yet, and also there are big gap in fermentative prodn level and international most advanced level.Enzyme preparation product is the mixing prod of multiple AMS, saccharifying enzyme and glucuroide, often can't effectively carry out the generation of purpose product.
Summary of the invention
The purpose of this invention is to provide a kind of AMS and application thereof, promptly a kind of novel AMS with industrial application value is to remedy the deficiency of prior art.
One aspect of the invention relates to a kind of AMS, includes:
A) sequence is the enzyme of SEQ ID NO:1;
B) aminoacid sequence in a) is through replacing, lack or adding one or several amino acid and have the active of enzyme described in a, by a deutero-enzyme.
The encode Nucleotide of above-mentioned AMS, its a kind of sequence is SEQ ID NO:2.
The present invention also is provided for expressing the recombinant expression plasmid of above-mentioned AMS, is to be that the nucleotide fragments of SEQ ID NO:2 is inserted in the prokaryotic expression carrier and makes up with sequence.
The invention still further relates to the reorganization bacterium that carries the recombinant expression plasmid of expressing above-mentioned AMS.
The AMS that the present invention filters out has been brought into play crucial effect in starch is hydrolyzed to the process of SANMALT-S and oligosaccharide and a small amount of glucose; Can be used for producing high maltose syrup, be used for the production of baked goods such as bread as additive; And application is in various degree all arranged in industries such as beer, yellow rice wine and raw material alcohol industries, have the industrial production using value.And the AMS that filters out can be used for transforming engineering bacteria, thereby obtains to have express alpha-diastatic reorganization bacterium.
Description of drawings
Fig. 1: be used to express the pGm-Pamy plasmid map of AMS of the present invention,
Fig. 2: the SDS-PAGE gel figure of AMS of the present invention;
Fig. 3: the pH stability of AMS of the present invention in the black mold host;
Fig. 4: AMS of the present invention is in the temperature stability in the black mold host.
Embodiment
Below in conjunction with instance method of the present invention is further specified.But instance only limits to explanation, is not limited to this.The experimental technique of unreceipted actual conditions in the following example usually can be by normal condition, the condition described in " the molecular cloning experiment guide " write like J. Sa nurse Brooker (Sambrook) etc., or the condition operation of advising according to manufacturer.
1, AMS Pamy's is synthetic
From the protein sequence that penicillium funiculosum (Penicillium Funiculosum) genome sequencing obtains, find penicillium funiculosum glycase (Penicillium Funiculosum amylase) sequence.With this sequence blast in NCBI; With known array alpha-amylase; The highest similarity of putative [Penicillium marneffei ATCC 18224] is 86%; The protein sequence of penicillium funiculosum glycase (Penicillium Funiculosum amylase) is carried out codon optimized and translates into corresponding nucleic acids sequence Pamy, Pamy is not cut off by Xho I and two restriction enzyme sites of Xba I, add Xho I and Xba I restriction enzyme site at Pamy nucleotide sequence two ends; It is synthetic that text sequence is delivered to biotech firm's (worker is given birth in Shanghai); Synthetic Pamy is connected on the carrier PSG-TS, and inserting the site is the t-a clone, and recipient bacterium is an e.colistraindh5.Wherein the nucleotides sequence of AMS of the present invention is classified SEQ ID NO:2 as, and aminoacid sequence is SEQ ID NO:1.
2, the structure of AMS Pamy gene recombined vector
With being connected in Pamy gene on the PSG-TS carrier,, cut glue and reclaim enzyme and cut the product fragment through agarose gel electrophoresis with Xho I and Xba I double digestion., behind transformed competence colibacillus intestinal bacteria (E.coli) the DH5 α, be applied on the LB solid medium that contains Amp with Xho I and Xba I double digestion and cut the plasmid pGm that glue reclaims and be connected with same.Cultivated 14-16 hour for 37 ℃; Carry out bacterium colony PCR checking, correct band is arranged, the picking mono-clonal is transferred in the 4ml LB liquid nutrient medium and cultivates (containing Amp); Cultivate after 14-6 hour; Extract plasmid, change recombinant plasmid pGm-Pamy over to expressive host black mold G1, contain the recombinant bacterial strain called after G1-pGm-Pamy of this recombinant plasmid.
Enzyme wherein cut and ligation all with reference to table 1.
Table 1 enzyme is cut system and linked system
3, transforming black mold and recombinant screen identifies
The 250ml4-flask with indentation that (1) 100ml CMA will be housed is with fresh black mold G1 bacterial strain spore inoculating, under 30 ℃, 200rpm condition, makes culture growth 12 hours.
(2) thalline that obtains in (1) is filtered with 4 layers of sterile gauze, first with aseptic water washing 3 times, wash 3 times with solution A again.
The mycelium that (3) under aseptic condition, will clean is transferred in the protoplastis solution and in 30 ℃, 200rpm incubation 2 hours, microscopic examination monitoring protoplast formation progress.
(4) with aseptic Micra-Cloth protoplast formation reaction filter is gone into to the aseptic disposable centrifuge tube of two 50ml, and the volume of every pipe is settled to 45ml with solution B, the centrifugal 10min of 4000rpm.Abandon supernatant; Xiang Guanzhong adds the 20ml solution B, mixing, and the centrifugal 5min of 4000rpm abandons supernatant; In pipe, add the 20ml solution B again, mixing, the centrifugal 5min of 4000rpm abandons supernatant.
(5) add the resuspended protoplastis of 100 μ l solution B, the concentration of protoplastis should reach 1 * 10
7Individual/100 μ l.On ice, 100 μ l protoplastis solution are transferred in the 15ml pipe of precooling each conversion reaction one pipe.In pipe, add 10 μ g DNA with the volume that is no more than 10 μ l, and add 12.5 μ l solution C, gentle mixing and incubation on ice 20 minutes.
(6) MMSA upper strata substratum (every pipe 8ml) is melted and remain on 55 ℃.From ice, shift out protoplastis, in protoplastis, add 1ml solution C and 2ml solution B, and with a pipe MMSA upper strata substratum gentleness mixing, with mixed solution pour into respectively in three amds lower floor culture medium flat plates 30 ℃ constant temperature culture 4-7 days.
(7) transformant that grows is forwarded to adms secondary checking culture medium flat plate; 30 ℃ of constant temperature culture, and carry out bacterium colony PCR checking, the positive strain of acquisition is identified correct through order-checking; The nucleotides sequence of AMS is classified SEQ ID NO:1 as, and its amino acid sequence coded is SEQ ID NO:2.
Solution A (every 500ml)-2.5ml 1M K
2HPO
4; 2.5ml 1M KH
2PO
4; 48.156g anhydrous MgSO
4(FW 120.37); Add dlH
2O is to final volume 500ml, pH5.5.With solution filtration sterilization and preservation at room temperature.
Protoplast formation solution-with callose enzyme (the InterSpex Products of 0.6g; Inc, CA) or 450mg callose enzyme and 150mg Driselase (InterSpex Products Inc.) is dissolved in the 40ml solution A; And with the solution filtration sterilization, 0.2 micron.
Solution B (every 500ml)-5ml 1M Tris, pH7.5; 2.77g CaCl
2(FW 110.99); 109.32g (FW 182.2 for sorbyl alcohol; 1.2M); Add dlH
2O is to final volume 500ml.With this solution filtration sterilization and preservation at room temperature.
Solution C (every 500ml)-250g PEG4000; 2.77g CaCl
25ml 1M Tris, pH7.5; Add dlH
2O is to final volume 500ml.With this solution filtration sterilization.
MMSA agar-with the 0.59g/L ethanamide; 3.4g/L CsCl; 0.52g/L KCl; 1.52g/L KH
2PO
4218.5g/L D-sorbyl alcohol; The 1ml/L trace elements; 10g/L agar (low melting-point agarose in the top-agar) is dissolved in 1L dlH
2O, autoclaving.After the sterilization, under aseptic condition, add 10ml50% glucose and 1.25ml 20% MgSO
47H
2O.
CMA (1 liter)=20g dextrose anhydrous; 20g malt extract (Difco Brand Malt Extract), 1g peptone (Bacto Peptone); 20g agar (Bacto Agar)
4, the determination of activity of αDian Fenmei
The positive strain that obtains is inoculated in the liquid fermentation medium, in 30 ℃, the cultivation of 200rpm shaking table.The αDian Fenmei enzyme is applied flexibly Megazyme Alpha-amylase Ceralpha method (Megazyme International Ireland) kit measurement.The glycase enzyme work that the black mold G1 that has changed the pGm-Pamy plasmid over to measures under pH5.4,40 ℃ of conditions can reach 135.64CU/ml.
4.1, measure the enzyme work of αDian Fenmei of the present invention under different pH:
Be set at pH2.4, pH3.0, pH3.6, pH4.2, pH4.8, pH5.4, pH6.0, pH6.6, pH7.2, pH7.8 respectively with surveying enzyme reaction buffer pH alive, the enzyme that is determined at αDian Fenmei under these pH conditions is lived, and measures the result referring to figure three.Can find out that from this figure the optimal reaction pH of αDian Fenmei is 6.6.
4.2, measure the enzyme of αDian Fenmei of the present invention under pH6.6, differing temps and live:
To react pH and be set at 6.6, the measured reaction temperature be set at respectively 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃.Measure the result referring to figure four.Can find out that from this figure the optimal reactive temperature of αDian Fenmei is 40 ℃.
5, the application example of AMS
Through the variation of research bread hardness and the relation between the AMS addition, find that AMS has certain antiageing effect.The result shows that in suitable interpolation scope (0.5～1.5ml/100g flour), AMS can increase the specific volume of bread significantly, reduces the hardness of bread shell and crumb, improves the bread quality, improves the quality that bakes of bread.AMS can improve the volume of bread, and the mouthfeel of improvement bread makes small product size such as bread bigger, and particle is more soft, and the quality that improves bread is had great significance.The present invention provides the certain theory foundation for AMS is applied to as bread additive in the bread industry.
Claims (6)
1. an AMS is characterized in that, described AMS includes:
A) sequence is the enzyme of SEQ ID NO:1;
B) aminoacid sequence in a) is through replacing, lack or adding one or several amino acid and have the active of enzyme described in a, by a deutero-enzyme.
2. a Nucleotide is characterized in that, the described Nucleotide described AMS of claim 1 that is used to encode.
3. Nucleotide as claimed in claim 2, the sequence that it is characterized in that described Nucleotide are SEQ ID NO:2.
4. a recombinant expression plasmid that is used to express the described AMS of claim 1 is characterized in that, described recombinant expression plasmid is to be that the nucleotide fragments of SEQ ID NO:2 is inserted in the prokaryotic expression carrier and makes up with sequence.
5. a reorganization bacterium is characterized in that described reorganization bacterium carries the described recombinant expression plasmid of claim 4.
6. the described AMS of claim 1 is as the application of bread additive.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849608A (en) * | 2013-12-10 | 2014-06-11 | 青岛蔚蓝生物集团有限公司 | High temperature-resistant amylase |
| CN104480027A (en) * | 2015-01-04 | 2015-04-01 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger strain of high-yield protease K and application of aspergillus niger strain |
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| WO2005117756A2 (en) * | 2004-05-27 | 2005-12-15 | Genencor International, Inc. | Acid-stable alpha amylases having granular starch hydrolyzing activity and enzyme compositions |
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|---|---|---|---|---|
| WO2005117756A2 (en) * | 2004-05-27 | 2005-12-15 | Genencor International, Inc. | Acid-stable alpha amylases having granular starch hydrolyzing activity and enzyme compositions |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849608A (en) * | 2013-12-10 | 2014-06-11 | 青岛蔚蓝生物集团有限公司 | High temperature-resistant amylase |
| CN103849608B (en) * | 2013-12-10 | 2016-09-07 | 青岛蔚蓝生物集团有限公司 | A kind of fire resistant alpha-diastase |
| CN104480027A (en) * | 2015-01-04 | 2015-04-01 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger strain of high-yield protease K and application of aspergillus niger strain |
| CN104480027B (en) * | 2015-01-04 | 2016-09-21 | 青岛蔚蓝生物集团有限公司 | The Aspergillus niger strain of a kind of high proteinase yield K and application thereof |
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| Publication number | Publication date |
|---|---|
| CN102827816B (en) | 2013-09-25 |
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