CN104862292B - A kind of new alpha amylase and its application - Google Patents
A kind of new alpha amylase and its application Download PDFInfo
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- CN104862292B CN104862292B CN201510205474.4A CN201510205474A CN104862292B CN 104862292 B CN104862292 B CN 104862292B CN 201510205474 A CN201510205474 A CN 201510205474A CN 104862292 B CN104862292 B CN 104862292B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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Abstract
It is an object of the invention to provide a kind of new alpha amylase, its amino acid sequence is SEQ ID NO：1, the gene of above-mentioned alpha amylase is encoded, one kind nucleotide sequence is SEQ ID NO：2.The alpha amylase of the present invention has broader temperature tolerance range, and specific activity of enzyme is measured at 50 DEG C and is up to 5776U/mg, and it remains to keep 38% or so activity at 10 DEG C of low temperature.
Description
Technical field
The invention belongs to functional gene triage techniques field, and in particular to a kind of Novel α amylase and its application.
Background technology
α-Isosorbide-5-Nitrae glycosidic bond inside the specific catalyzing hydrolysis starch molecule of alpha-amylase, Starch Hydrolysis can be by it
Smaller fragment.Its main hydrolase as degradable starch, alpha-amylase constitute a very huge glycosyl hydrolase
Family.The amylase having now been found that largely has acidproof, resistant to elevated temperatures property, although many species can produce amylase,
But the alpha-amylase of business application is essentially from bacillus (bacillus licheniformis, bacillus stearothermophilus reconciliation shallow lake
Afnyloliquefaciens etc.).Extensive commercially available heat-resistant alpha-amylase is essentially from bacillus subtilis, stearothermophilus at present
Bacillus, bacillus licheniformis and bacillus amyloliquefaciens.But rarely have the industrial applications of the high alpha-amylase living of low temperature
Report.And the alpha-amylase of high vigor is kept under low temperature to having the technique of distinct temperature requirement and saving energy etc. with very high
Application potential, so the high living Novel α amylase tool of screening low temperature is of great significance
The content of the invention
It is an object of the invention to provide a kind of Novel α amylase, so as to make up the deficiencies in the prior art.
The alpha-amylase of the present invention, includes
1) amino acid sequence is SEQ ID NO：1 alpha-amylase；
2) 1) substituted, lack, added it is one or several amino acids formed, and the α with zymologic property in 1)-
Amylase；
The gene of above-mentioned alpha-amylase is encoded, one kind nucleotide sequence is SEQ ID NO：2；
The present invention also provides a kind of recombination bacillus coli, and conversion has the expression plasmid for carrying said gene.
Above-mentioned recombination bacillus coli, it is Escherichia coli XH031-A1 (Escherichia coli XH031-A1), in
On March 25th, 2015 China typical culture collection center for being preserved in Wuhan, China Wuhan University, deposit number CCTCC
NO:M2015163。
The alpha-amylase of the present invention has broader temperature tolerance range, and measuring specific activity of enzyme at 50 DEG C is up to 5776U/
Mg, and the activity that it remains to keep 38% or so at 10 DEG C of low temperature.
Brief description of the drawings
Fig. 1：The graph of a relation of the activity and temperature of the alpha-amylase of the present invention.
Embodiment
Embodiment 1：Alpha-amylase gene laxh3357 acquisition
Analyzed by carrying out full-length genome to the new bacterium XH031 in deep-sea, obtain 3 amylase genes and its gene sequence
Row, sense primer (5 '-CGGAATTCATGCACCCTCGACCGGC-3 ') and downstream are designed using biological software Primer5.0
Primer (5 '-CCCTCGAGACGCCGCCACATCCG-3 ') enters performing PCR reaction, PCR reaction compositions using genomic DNA as template
(50 μ l reaction systems) as follows：ddH2μ l of O 11.5, upstream and downstream primer each 0.5 μ l, μ l of DNA profiling 2,2 × GC
Buffer25μl、Taq 0.5μl、dNTP Mixture 8μl.Reaction condition is：95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 30s,
65 DEG C of annealing 30s, 72 DEG C of extension 3min30s, 72 DEG C extend 10min eventually, totally 30 circulations.After reaction terminates, PCR primer is returned
Receipts obtain alpha-amylase gene laxh3357.Laxh3357 is one of alpha-amylase gene, and its nucleotides sequence is classified as SEQ
ID NO：2, the amino acid sequence of coding is SEQ ID NO：1, with its most like albumin A lpha-amylase
(Streptomyces hygroscopicus), entry：P08486, identity are only 28.3%.Before showing that it is one kind
The alpha-amylase gene do not reported.
Embodiment 2：E. coli cloning vector PUCm-T-laxh3357 structure.
Alpha-amylase gene laxh3357 and carrier PUCm-T connection, using DNA Ligation Kit connections, connection
System (10 μ l) is as follows：SolutionI5 μ l, the μ l of 4 μ l, PUCm-T carrier of DNA fragmentation 1.Connection obtained by 16 DEG C of connection 16h
Liquid can obtain E. coli cloning vector PUCm-T-laxh3357, for converting e. coli jm109.
Embodiment 3：Escherichia coli recombinant strain JM109-PUCm-T-laxh3357 structure
Add 200 μ l defrostings competence Ecoli.JM109 and the obtained connection liquid of 10 μ l embodiments 2 extremely
In 2mlEppendorf pipes, ice bath 30min, 42 DEG C of heat shock 90s, ice bath 15min, the μ l of LB culture mediums 800,37 DEG C of vibrations are added
Cultivate 45min.Bacterium solution is coated on the LB containing 100 μ g/ml ampicillins after being mixed with 4 μ l IPTG and 40 μ l X-gal and put down
On plate, 37 DEG C of culture 12-14h.Picking white colony is PCR and double digestion detection, digestion system (20 μ l) are as follows：ddH2O 8μ
The μ l of 8 μ l, EcoRI1 μ l, XholI1 μ l, 10 × H Buffer of l, PUCm-T-laxh3357 DNA 2, Ago-Gel electricity
It is positive transformants clone to occur the special band persons of 1428bp in swimming, i.e., the e. coli jm109 containing PUCm-T-laxh3357-
PUCm-T-laxh3357.1ml positive colony bacterium solutions are sent to survey, with M13 sequencing universal primer measure external source insetion sequences
Laxh3357 nucleotide sequences.
Embodiment 4：Coli expression carrier pET24a (+)-laxh3357 structure
Cloning vector PUCm-T-laxh3357 and expression vector pET24a (+) uses EcoRI and XholI double digestions, enzyme respectively
It is as follows to cut system (20 μ l)：
Reaction system 1：ddH28 μ l, PUCm-T-laxh3357 DNAs of O 8 μ l, EcoRI1 μ l, XholI1 μ l, 10 ×
The μ l reaction systems 2 of H Buffer 2：ddH2O 8 μ l, pET24a (+) DNA 8 μ l, EcoRI1 μ l, XholI1 μ l, 10 × H
Buffer 2μl.
37 DEG C of digestion 30min, two purpose fragments 1428bp and 5.3Kb are separately recovered after electrophoresis, the two respectively with α-shallow lake
Powder enzyme YM01 full length genes fragment is identical with expression vector PET24a (+) clip size, with DNA Ligation Kit connections, connects
Junctor system (10 μ l) is as follows：SolutionI5 μ l, DNA fragmentation 1.5 μ l, pET24a (+) carrier 1.1 μ l, ddH2O 2.4μl。16
DEG C connection can obtain coli expression carrier pET24a (+)-laxh3357 in 16 hours, for converting e. coli bl21
(DE3)。
Embodiment 5:Escherichia coli restructuring BL21 (DE3)-pET24a (+)-laxh3357 (XH031-A1) structure
Add 200 μ l defrostings competence E.coliBL21 and the obtained connection liquid of 10 μ l embodiments 4 extremely
In 2mlEppendorf pipes, ice bath 30min, 42 DEG C of heat shock 90s, ice bath 15min, the μ l of LB culture mediums 800,37 DEG C of vibrations are added
Cultivate 45min.Bacterium solution is coated on the LB flat boards containing 100 μ g/ml kanamycins, 37 DEG C of culture 8-12h.Picking colony does double
Digestion detects, and digestion system (10 μ l) is as follows：ddH2The μ l, EcoRI 0.5 of O 4 μ l, pET24a (+)-laxh3357 DNAs 4
The μ l of 0.5 μ l, 10 × H Buffer of μ l, XhoI 1.It is positive transformants gram to occur the special band persons of 1428bp in agarose gel electrophoresis
It is grand, i.e., Escherichia coli recombinant strain BL21 (DE3)-pET24a (+)-laxh3357 containing pET24a (+)-laxh3357.
Embodiment 6：The preparation of recombinant alpha-amylases
Picking Escherichia coli recombinant strain BL21 (DE3)-pET24a (+)-laxh3357 monoclonals are in being 100 μ containing concentration
In the LB of g/ml kanamycins fluid nutrient medium, 37 DEG C of shaken cultivations are stayed overnight, and being seeded to 200ml by 1% inoculum concentration contains 100
In the LB of μ g/ml kanamycins fluid nutrient medium, 37 DEG C of 150rpm shaken cultivations to OD600For 0.4-0.5, final concentration is added
For 0.1mMIPTG, 16 DEG C of induced expression 16-20h.4 DEG C of 6000rpm centrifugation 10min of bacterium solution are induced, collect thalline, thalline is used
10ml1 × Ni posts combination buffer (20mM Tris-HCl, PH=8.0；10mM imidazoles；0.5mM NaCl) it is resuspended and is surpassed
Sonication, 4 DEG C of 12000rpm centrifugation 10min, collect supernatant after crushing.Supernatant up to uses the Ni posts balanced, with 10
Times volume 1 × Ni posts lavation buffer solution (20mM Tris-HCl, PH=8.0；20mM imidazoles；0.5mM NaCl) elution, Ran Houyi
The secondary lavation buffer solution washing affinity column with various concentrations (20mM, 50mM, 150mM) imidazoles, it is finally 250mM imidazoles with concentration
Eluent washing affinity column, collect eluent.Eluent is in dialyzate (20mM Tris-HCl, PH=8.0；0.85(g/v)
NaCl, 10% glycerine (v/v)) in dialysis 24h after SDS-PAGE detection eluent in albumen be distributed, merge band containing single goal
Eluent i.e. can obtain recombinant alpha-amylases.With Bradford methods measure destination protein concentration, after packing by albumen freeze in-
20 DEG C preserve with standby.
Embodiment 7：Recombinant alpha-amylases Activity determination and its zymologic property
Punched on amylase flat board, take the blank control supernatant coli strain XH031- of 10 microlitres of inductions respectively
Check on corresponding aperture, 37 DEG C of placement 3h, then dyed with Lugol's iodine solution on A1 albumen, as a result can be in brown background
There is obvious transparent circle to XH031-A1, then find to have on starch plate by the recombinase same method point of purifying larger transparent
Circle, it was demonstrated that its activity is high.
The zymologic property that this agarase is measured with DNS reagent methods is as follows：
1st, optimum temperature be that enzyme activity is most suitable enzyme activity at 50 DEG C, 10 DEG C 38%.
2nd, heat endurance：1h is placed at 0 DEG C -100 DEG C, wherein 0 DEG C -45 DEG C activity that can keep 100%.
3rd, optimal pH 10.0
4th, PH stability：12h is placed in the buffer solution of PH=3~12, wherein PH=6-11 can keep more than 57% activity.
5th, influence of the different ions to enzyme activity：Metal ion such as Na+、K+、Mn2+The work of enzyme can be improved in low concentration 1mM
Power, it is respectively increased 21.5%, 8.1% and 17.2%；Heavy metal ion such as Fe3+、Zn2+、Ag+There is obvious suppression to make to enzyme activity
With.
6th, enzyme is than living：Specific activity of enzyme is measured at 50 DEG C and is up to 5776U/mg (Fig. 1)
The high vigor alpha-amylase of low temperature of the present invention is in detergent industry, it is not necessary to which higher water temperature is with regard to that can reach good
Clean result, so as to save the energy；In food industry, the fermentation time of dough/pasta can be shortened, improve the quality of dough/pasta.
More important point, the amylase can adapt to wider temperature range, be applicable to the technological process of temperature change, such as
In terms of feed addition, feed preparation needs higher temperature, and 37 degrees Celsius of the greenhouse cooling entered in letting animals feed body, whole mistake
The journey enzyme can play good effect.
Claims (8)
1. a kind of alpha-amylase, it is characterised in that the amino acid sequence of described alpha-amylase is SEQ ID NO：1.
A kind of 2. gene, it is characterised in that the alpha-amylase described in described gene code claim 1.
3. gene as claimed in claim 2, it is characterised in that the nucleotides sequence of described gene is classified as SEQ ID NO：2.
4. a kind of recombinant plasmid, it is characterised in that described recombinant plasmid is used to recombinantly express the alphalise starch described in claim 1
Enzyme.
5. recombinant plasmid as claimed in claim 4, it is characterised in that described recombinant plasmid is carried described in claim 2
Gene.
A kind of 6. recombination bacillus coli, it is characterised in that the restructuring that described recombination bacillus coli conversion is had the right described in requirement 4
Plasmid.
7. recombination bacillus coli as claimed in claim 6, it is characterised in that the deposit number of described recombination bacillus coli is
CCTCC NO:M2015163。
8. application of the alpha-amylase described in claim 1 in field of feed processing.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510205474.4A CN104862292B (en) | 2015-04-25 | 2015-04-25 | A kind of new alpha amylase and its application |
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| CN201510205474.4A CN104862292B (en) | 2015-04-25 | 2015-04-25 | A kind of new alpha amylase and its application |
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| CN104862292A CN104862292A (en) | 2015-08-26 |
| CN104862292B true CN104862292B (en) | 2017-11-10 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500871A (en) * | 2002-11-19 | 2004-06-02 | 中国科学院微生物研究所 | High temperaturealpha- amylase and coding gene thereof |
| CN1500870A (en) * | 2002-11-19 | 2004-06-02 | 中国科学院微生物研究所 | Alkalinealpha- amylase, coding gene and uses thereof |
| CN1952115A (en) * | 2005-10-19 | 2007-04-25 | 新疆农业科学院微生物应用研究所 | Low-temperature amylase strain, low-temperature amylase, and production therefor |
-
2015
- 2015-04-25 CN CN201510205474.4A patent/CN104862292B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500871A (en) * | 2002-11-19 | 2004-06-02 | 中国科学院微生物研究所 | High temperaturealpha- amylase and coding gene thereof |
| CN1500870A (en) * | 2002-11-19 | 2004-06-02 | 中国科学院微生物研究所 | Alkalinealpha- amylase, coding gene and uses thereof |
| CN1952115A (en) * | 2005-10-19 | 2007-04-25 | 新疆农业科学院微生物应用研究所 | Low-temperature amylase strain, low-temperature amylase, and production therefor |
Non-Patent Citations (3)
| Title |
|---|
| LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T;XH031TQinghao Song,et al;《Enzyme and Microbial Technology》;20160509;第90卷;83－92 * |
| Luteimonas abyssi sp. nov., isolated from deep-sea sediment;Xiaoyang Fan,et al;《International Journal of Systematic and Evolutionary Microbiology》;20141231;第64卷;668－674 * |
| 南太平洋沉积物深渊藤黄单胞菌 (Luteimonas abyssi sp.nov.) 等4 个深海细菌新种的分类鉴定;范晓阳;《中国优秀硕士学位论文全文数据库 基础科学辑》;20150115(第1期);A006-284 * |
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