CN103834606A - Engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants - Google Patents
Engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants Download PDFInfo
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Abstract
The invention discloses an engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants. The engineering strain is obtained by introducing a recombinant vector expressing acid-resistant high-temperature alpha-amylase genes into bacillus licheniformis, and performing screening to obtain the engineering strain with improved acid-resistant high-temperature alpha-amylase expression level; the acid-resistant high-temperature alpha-amylase has an amino acid sequence as shown in sequence 2 in the sequence table. The engineering strain has stronger capability of producing acid-resistant high-temperature alpha-amylase than current domestic production strains, has a unique identification label, and is a bacillus licheniformis production strain of acid-resistant high-temperature alpha-amylase that has great production and application value.
Description
Technical field
The present invention relates to a kind of engineering strain of expressing acid resistance high-temperatureα-amylase gene.Be by chemical mutagen, high-temperatureα-amylase gene amy to be suddenlyd change specifically, then screen and obtain novel acidproof high-temperatureα-amylase gene mutation body amyM by acidic conditions, in Bacillus licheniformis engineering bacteria, realize and expressing and application.
Background technology
High-temperatureα-amylase, can hydrolyzed starch, a-1 in soluble dextrins and oligose, and the α-amylase of 4 glucoside bonds, because its optimal reactive temperature is higher, is generally more than 60 degrees Celsius, is therefore called high-temperatureα-amylase.
High-temperatureα-amylase is widely used in the industrial production such as drinks, monosodium glutamate and β-amylose, and replacement traditional technology both saving of labor is economized plant and equipment, can improve again yield rate, reduces costs, and increases benefit.B.lieheniformis α-amylase is the widest efficient liquefying amylase of one of the maximum purposes of China's output, it is by a kind of zymin that after the high B.lieheniformis mutagenesis of resistance toheat, enlarged culturing obtains through purification more step by step, and Main Function is to be dextrin by the starchiness liquefaction in raw material.
At present, diastatic optimum pH conventional in domestic and international market is 6.0～10.0, and under acidic conditions, its enzymic activity reduces clearly, even loses activity.Along with the development of China's starchy material deep processing industries, the continuous change of processing condition, some starch material deep processing technology need to carry out under lower pH value condition.So development of new acid-resistant alpha-amylase is extremely urgent.
Owing to screening the wild strain that obtains from nature, to produce the vigor of acid alpha-amylase generally all lower, can not directly apply to industrialized fermentative production.Chinese scholars, generally by the technique means such as mutagenesis, cross-breeding to wild strain, improves the enzymatic productivity of bacterial strain, and Qi Wei etc. are by utilizing low energy N
+inject subtilis, the bacterial strain obtaining after mutagenesis, the enzyme of its acid alpha-amylase is lived as 343U/ml, after mutagenesis, compare with starting strain, Optimun pH is on the low side unit (Qi Wei, a Wang Haiyan, Wang Jianling, etc. the research that the ion implantation seed selection acid resistance of nitrogen α 2 amylase produce bacterium mutagenic effect.University Of Science and Technology Of Tianjin's journal, 2007,22 (9): 7-8.).Develop rapidly along with molecular biological, genetic engineering breeding technology is owing to can gene being designed in advance and be controlled, and expressional function successful, so be more and more subject to domestic and international investigator's favor.Cai is identical with after the genetic modification of α-amylase, be cloned in shuttle plasmid pBE2, build the secretor type inducible expression carrier pBSAT containing mutant alpha-amylase gene, the bacterial strain that contains pBSAT can be secreted into mutant alpha-amylase outside born of the same parents, the α-amylase of secretion has higher acid resistance (Cai Heng, Chen Zhongjun, Li Jinxia, etc.Acid resistance transformation and the secreting, expressing in subtilis of α-amylase.Food and fermentation industries, 2005,31 (10): 33-36.), European Studies person adopts genetic modification Bacillus licheniformis to produce stable on heating acid alpha-amylase, in the DNA sequence dna of original natural bacterial strain, the aspartic acid of N188 position is replaced with other any amino acid, make the methionine residue disappearance in N15 or N197 position, the applicable liquefaction pH value of α-amylase that the superior strain obtaining is produced can be reduced to 5.0, and constant (the SHAW A of activity in the time of 100～110 ℃, BOTT R, DAY A G.Protein engineering of α-amylase for low pH performance.Curr Opin Biotechnol, 1999 (10): 349-352.).
China is large agricultural country, and starch resource is very abundant, and the range of application of acid starch enzyme is also more and more extensive.As far back as 20 century 70s, domestic just have scholar to start research to acid starch enzyme, obtained some achievements in research, but compared with abroad, gap is very far away, and the bacterial strain that China produces acid alpha-amylase is more single, and enzyme is lived lower.
Summary of the invention
The object of this invention is to provide a kind of engineering strain of expressing acid resistance high-temperatureα-amylase.
Engineering bacteria of expressing high-temperatureα-amylase provided by the present invention, will come from Bacillus licheniformis (Bacillus Licheniformis) high-temperatureα-amylase gene to suddenly change, obtain mutant, then import in Bacillus licheniformis the engineering strain that the high-temperatureα-amylase acid resistance that screening obtains improves expressing acid resistance high-temperatureα-amylase gene mutation body recombinant vectors; Described high-temperatureα-amylase aminoacid sequence is as shown in sequence table 1; Described acid resistance high-temperatureα-amylase mutant aminoacid sequence is as shown in sequence table 2.
Described Bacillus licheniformis (Bacillus Licheniformis) is Bacillus licheniformis (Bacillus Licheniformis) CICC10181.Described engineering bacteria of expressing acid resistance high-temperatureα-amylase is preferably Bacillus licheniformis (Bacillus Licheniformi5) ZHWY CGMCC No.6669.
Described Bacillus licheniformis (Bacillus Licheniformis) ZHWY CGMCC No.6669, be preserved in Chinese microorganism strain preservation board of trustee reason person on October 12nd, 2012 and understand common micro-organisms center (abbreviation CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), preserving number is CGMCC No.6669.
Described fermentation is the Semen Maydis powder containing 75-86g/L with substratum, the soybean cake powder of 35-45g/L, the calcium chloride of 1-3g/L, the Sodium phosphate dibasic of 6.5-8.5g/L, the liquid nutrient medium of the ammonium sulfate of 3.5-5.5g/L.
The temperature of described fermentation is 36.0-38.5 ℃, is preferably 37 ℃.
Engineering bacteria of expressing acidproof high-temperatureα-amylase of the present invention, particularly Bacillus licheniformis (Bacillus Licheniformis) ZHWY CGMCC No.6669, have than current domestic high-temperatureα-amylase and produce the stronger acid-fast ability of bacterial strain, there is unique appraisement label, be a strain have production application be worth acidproof high-temperatureα-amylase Bacillus licheniformis produce bacterial strain.Experiment showed, under identical fermentation condition, the optimal pH of the high-temperatureα-amylase that the engineering strain that contains acidproof alpha-amylase gene mutation body of the present invention is produced is 5.2, and the optimal pH of the high-temperatureα-amylase of wild type strain is 6.4.
Accompanying drawing explanation
Fig. 1 is the structure iron of pAXOI-amyM.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions.
Embodiment 1, the mutagenic obtained acid resistance high-temperatureα-amylase of Sodium Nitrite mutant gene amyM
Extract the genomic dna of Bacillus licheniformis CICC10181 (being purchased from Chinese industrial microbial strains preservation administrative center).(extracting method is with reference to Bron, S (1990) .Plasmids.In Molecular Biological Methods for Bacillus, pp75-174.Edited by C.R.Harwood & S.M.cutting.Chichester:Wiley).
Carry out pcr amplification amy gene take Bacillus licheniformis (B.Licheniformis) CICC10181 genomic dna as template, upstream primer is that primer 1 (5 '-ACCTGCCTGTACACTTGCG-3 ') and downstream primer are primer 2 (5 '-CTCTCTGCTCTTCTATCTTTG-3 ').
Amplification condition: 94 ℃ of denaturations 5 minutes, then 94 ℃ of sex change 1 minute, anneal 56 ℃ one minute, extend 72 ℃ 1 point 30 seconds.Complete after 30 circulations, 72 ℃ are incubated 10 minutes.
Above-mentioned amplification obtains the amy fragment of 1978bp, shows through order-checking, and this fragment comprises promotor, signal coding sequence and the structure gene of high-temperatureα-amylase gene.This DNA fragmentation coding aminoacid sequence as shown in sequence in sequence table 1.
Amy gene is connected with coli expression carrier pEASY-T3, and Transformed E .coli Top10, extracts plasmid pEASY-T3-amy after cultivation enrichment.
The plasmid pEASY-T3-amy extracting is carried out to Sodium Nitrite mutagenesis and screening obtains pEASY-T3-amyM, and method is as follows:
1. in EP pipe, add 1.0 μ gDNA.
2. the Sodium Nitrite 200 μ L that add 50mMol/L, 100mMol/L sodium-acetate buffer (pH4.6) 200 μ L, mix, and leave standstill 15-60 minute at 20 ℃.
3. add 100 μ L2.5Mol/L sodium-acetate buffers (pH7.0) to mix, stop Sodium Nitrite mutagenesis reaction.
4. add 2 times of cold ethanol of volume 100%, mix, place 30 minutes for-20 ℃.
5. centrifugal 10 minutes of 12000rpm, removes supernatant liquor, and precipitation is dried, and adds appropriate amount of deionized water to dissolve.
6. the plasmid that contains mutator gene is transformed and enters E.coli Top10 competent cell, in LB liquid nutrient medium, cultivate.
7. the intestinal bacteria of cultivating 12 hours are coated to the LB substratum of different pH values (pH5.0-5.8).The size of observing its growing state and producing transparent circle, is chosen in growth and the larger bacterium colony of transparent circle on lower pH flat board, extracts plasmid and checks order, and obtains acid resistance high-temperatureα-amylase mutant gene amyM.
Analyze according to amino acid code that amyM nucleic acid sequencing result is corresponding, result shows, compared with the wild-type high-temperatureα-amylase of amy coding, there is following variation: the lysine mutation of the 4th is l-asparagine in the acid resistance high-temperatureα-amylase mutant of amyM coding; The 134th arginine sports leucine; The asparagine mutation of the 288th is aspartic acid; The phenylalanine of the 289th sports tyrosine; The alanine mutation of the 320th is Serine.Acid resistance high-temperatureα-amylase mutant aminoacid sequence is as shown in sequence table 2.
The structure that carries LacA integration arm shuttle vectors pAXOI-amyl of embodiment 2, expression acid resistance high-temperatureα-amylase mutant gene amyM
Carry out pcr amplification acid resistance high-temperatureα-amylase mutant gene amyM take plasmid pEASY-T3-amyM as template.Amplimer is: upstream primer 5 '-cg
ggatccaCCTGCCTGTACACTTGCG-3 ' (underscore mark part is BamHI restriction enzyme site) and downstream primer 5 ' tccat
ccgcgg-CTCTCTGCTCTTCTATCTTTG-3 ' (underscore mark part is SacII, restriction enzyme site).
Above-mentioned amplification obtains the amyM fragment of 1978bp, shows through order-checking, and this fragment comprises promotor, signal coding sequence and the structure gene of acid resistance high-temperatureα-amylase gene.
Respectively with BamHI and SacII double digestion PCR product amyM and the integrated plasmid pAXOI that shuttles back and forth (purchased from preserving center, Bacillus Genetic Stock Center (BGSC) from Mei Wei Ohio State University bacterial classification
the Ohio State?
university), enzyme is cut to product and reclaim after test kit recovery purifying with PCR product respectively, adopt T4DNA ligase enzymes to carry out ligation 16 hours at 4 ℃.Ligation reaction is transformed to Host Strains E.coli Top10 (purchased from Beijing Tian Gen biochemical technology company limited) competent cell, and coating LB flat board (Amp+), selects positive colony, carries out shake-flask culture 16 hours.Harvested cell, extracts in a small amount plasmid and carries out that enzyme is cut and sequence verification.The expression that the correct shuttle plasmid of checking is carried to promotor, signal peptide and the structure gene of the acid resistance alpha-amylase integrated plasmid called after pAXOI-amyM (structure iron as shown in Figure 1) that shuttles back and forth.
The structure of the engineering bacteria ZHWY of embodiment 3, expression acid resistance high-temperatureα-amylase gene mutation body
The expression of the promotor of carrying acid resistance alpha-amylase, signal peptide and the structure gene that embodiment 2 mutagenesis the are obtained integrated plasmid pAXOI-amyM that shuttles back and forth adds electroporation (Romero D with protoplastis, Journal of Microbiology Methods, 2006Vol66p556-559) the Bacillus licheniformis CICC10181 of conversion product high-temperature starch (is purchased from Chinese industrial microbial strains preservation administrative center, preserving number is CICC10181), concrete grammar is as described below:
Adopt Romero D Bacillus licheniformis method for preparing protoplast (Romero D, Journal of Microbiology Methods, 2006Vol66p556-559) obtain after Bacillus licheniformis CICC10181 protoplastis, get plasmid pAXOI-amyM after 5ul (2ug) purifying in the centrifuge tube of a 1.5ml, the electric shock cup of itself and 0.2CM is placed on to precooling on ice, the protoplastis that 120ul is prepared proceeds to the centrifuge tube of the 1.5ml of the above-mentioned pAXOI-amyM of being equipped with, carefully mix, place 10min on ice, then open electroporation, adjust voltage to 600V, plasmid and protoplasm somatocyte mixed solution are moved on in the electric shock cup of precooling, dry electric shock cup with dry filter paper, note must not there be bubble in mixed solution.Electric shock cup is put into the cup groove of electroporation, press shock button, after having discharging sound to occur, in electric revolving cup, add 1ml cell recovery damping fluid (Romero D immediately, Journal of Microbiology Methods, 2006Vol66p556-559), after re-suspended cell, transfer in the centrifuge tube of 1.5ml.
The above-mentioned centrifuge tube that cell after conversion is housed is placed to 37 ℃, 100rpm shaking table is cultivated 12-16 hour, get 150ul bacterium liquid coat the DM3 solid medium that contains 10ug/ml erythromycin (containing 8g/L agar, 5g/L caseinhydrolysate, 5g/L yeast powder, 1.5g/L KH
2pO
4, 3.5g/L K
2hPO
4, the substratum of 45.5g/L sorbyl alcohol and 10g/L starch) flat board, put into Bacillus licheniformis appears in 37 ℃ of incubator Pei Raising single bacterium colony after 72-96 hour, picking list bacterium colony is can be at the positive colony containing growing on 10ug/ml erythromycin flat board, the positive strain obtaining is extracted to genomic gene, carry out PCR evaluation with the upstream and downstream primer of the gene (amy) of the amplification high-temperatureα-amylase described in embodiment 1, the bacterial strain that amplification obtains the fragment of 1978bp is positive strain, result obtains the gene recombination bacterium that a strain checking shows correct importing pAXOI-amyM, by this bacterial strain called after Bacillus licheniformis (Bacillus Licheniformis) ZHWY.This Bacillus licheniformis (Bacillus Licheniformis) ZHWY CGMCC No.6669, be preserved in Chinese microorganism strain preservation board of trustee reason person on October 12nd, 2012 and understand common micro-organisms center (abbreviation CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), preserving number is CGMCC No.6669.
Embodiment 4, the checking of gene recombination Bacillus licheniformis ZHWY acid resistance high-temperature starch expression of enzymes
Adopt 500 milliliters of triangular flasks with plate washer, the amount of dress substratum is 50ml, embodiment 2 gained Bacillus licheniformis (Bacillus Licheniformis) ZHWY or Bacillus licheniformis (B.Licheniformis) CICC10181 (wild mushroom) are inoculated in a triangular flask according to the amount of 0.01g thalline/ml substratum respectively, cultivate at 37 ℃ of shaking tables, shaking speed is 200rpm.Fermentation 96h, stops fermentation.Process for two groups and establish respectively three repetitions.
Consisting of of described substratum: soybean cake powder 26.1g/L, cottonseed meal 21.1g/L, calcium chloride 0.46g/L, ammonium sulfate 5g/L, Trisodium Citrate 2g/L, dipotassium hydrogen phosphate 14g/L, potassium primary phosphate 6g/L, corn steep liquor 21.1g/L, lactose 105g/L (independent sterilizing), pH value is 6.7, and all the other are water.
In fermenting process, get fermented supernatant fluid and carry out acid resistance detection and enzyme biopsy survey, method is as follows:
Get fermented liquid 1ul, point, to the 0.5% starch flat board of different pH (5.0-6.6, interval 0.2), reacts 15 minutes, measures the size of transparent circle.
Result shows, fermentation culture 96 hours, restructuring Bacillus licheniformis ZHWY transparent circle maximum on the starch flat board of pH5.2, and wild mushroom Bacillus licheniformis (B.Licheniformis) CICC10181 transparent circle maximum on the starch flat board of pH6.4 shows that acid resistance high-temperatureα-amylase gene mutation body amyM is stronger than wild-type alpha-amylase gene amy acid resistance.
Claims (5)
1. express the engineering bacteria of acidproof high-temperatureα-amylase, that the recombinant vectors that contains acid resistance high-temperatureα-amylase gene mutation body amyM is imported in Bacillus licheniformis (Bacillus Licheniformis), the engineering strain that the acidproof high-temperatureα-amylase expression amount that screening obtains improves.
2. engineering bacteria according to claim 1, is characterized in that: the sequence of the described acid resistance high-temperatureα-amylase mutant of described acid resistance high-temperatureα-amylase gene amyM coding is as shown in sequence table 2.
3. engineering bacteria according to claim 2, is characterized in that: described acid resistance high-temperatureα-amylase mutant including but not limited to: the lysine mutation of the 4th is l-asparagine; The 134th arginine sports leucine; The asparagine mutation of the 288th is aspartic acid; The phenylalanine of the 289th sports tyrosine; The alanine mutation of the 320th is Serine.
4. engineering bacteria according to claim 3, is characterized in that: described Bacillus licheniformis (Bacillus Licheniformis) is Bacillus licheniformis (Bacillus Licheniformis) ZHWY CGMCC No.6669.
5. expressing a method for acidproof high-temperatureα-amylase, is the engineering bacteria of the acidproof high-temperatureα-amylase of expression described in any one in fermentation claim 1-4, obtains acidproof high-temperatureα-amylase.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802940A (en) * | 2016-04-18 | 2016-07-27 | 广西大学 | Bacillus licheniformis high-temperature alpha-amylase mutant and application thereof |
| CN106086048A (en) * | 2016-08-16 | 2016-11-09 | 吉林大学 | A kind of acid resistance high temperature alpha amylase and gene, engineering bacteria and preparation method |
| CN106591261A (en) * | 2015-10-14 | 2017-04-26 | 中国科学院天津工业生物技术研究所 | Alpha amylase and mutant strain with high yield of alpha amylase |
| CN112391324A (en) * | 2020-12-09 | 2021-02-23 | 山东隆科特酶制剂有限公司 | Strain for high-yield acid-resistant high-temperature alpha-amylase 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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| CN106591261A (en) * | 2015-10-14 | 2017-04-26 | 中国科学院天津工业生物技术研究所 | Alpha amylase and mutant strain with high yield of alpha amylase |
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| 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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Application publication date: 20140604 |