CN1786172A - Thermophilic alkali alpha amylase and its coding gene - Google Patents
Thermophilic alkali alpha amylase and its coding gene Download PDFInfo
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- CN1786172A CN1786172A CN 200510116749 CN200510116749A CN1786172A CN 1786172 A CN1786172 A CN 1786172A CN 200510116749 CN200510116749 CN 200510116749 CN 200510116749 A CN200510116749 A CN 200510116749A CN 1786172 A CN1786172 A CN 1786172A
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Abstract
The invention relates to alpha-amylase and its coding gene. The enzyme has addiction heat alkalinity. Its optimum reaction temperature is about 60centigrade degree; its pH value is about 10. The enzyme can hydrolyze amylum to corresponding monose and oligosaccharide. It has important application value in ferment, weaving, medicine, foods, and so on.
Description
Technical field
The present invention relates to a kind of enzyme and encoding gene thereof, particularly a kind of thermophilic alkali alpha amylase and encoding gene thereof.
Background technology
Starch is to be made up of by α-1,4 or α-1,6 glycosidic link the α-Pu Taotang unit, is divided into amylose starch and amylopectin.Starch except that edible, industrial have many uses too general, as brewing alcoholic beverages by fermentation and refining sugar by hydrolysis.Because starch has complicated structure, degraded starch to need plurality of enzymes.These enzymes can be divided into 2 classes simply: excision enzyme and restriction endonuclease.Restriction endonuclease such as α-Dian Fenmei (EC3.2.1.1), excision enzyme such as beta-amylase (EC 3.2.1.2).
α-Dian Fenmei (α-amylase, EC 3.2.1.1) also claims liquefaction type amylase, α-1 in its hydrolyzed starch polysaccharide molecule such as amylose starch and the amylopectin, and the 4-glycosidic link produces monose and oligosaccharides.α-Dian Fenmei is used to a plurality of fields always for a long time.For example, it is used to the saccharification of cereal and potato always in fermentation industry, is used as the starch paste remover in textile industry, is used as digestants in pharmacy industry, and is used to make the heavy-gravity malt syrup in grocery trade.α-Dian Fenmei obtains from many different sources such as bacterium, fungi, plant and animal.
High-temperature has: (1) reduces starch wine with dregs viscosity, reduces the power consumption when carrying; (2) the living contaminants chance is few; (3) Heat stability is good, to advantages such as the needs of calcium ion are few, thereby at many production fields, during particularly Production by Enzymes glucose and high fructose syrup, alcohol and monosodium glutamate etc. were produced, high temperature resistant α-Dian Fenmei just progressively replaced the normal temperature α-Dian Fenmei.High-temperature mainly is to comprise warm bacterium (Takagi etc. from bacterium, Bacterial and Mold Amylase, The Enzymes, New York, Academic Press, 1971), moderate thermophile bacteria (Antranikian, AppliedBiochemistry and Biotechnology, 20/21:267-279,1989; Glymph etc., Appliedand Environmental Microbiology, 34:391,1977; Hasegawa etc., J.Biochem, 79:35-42,1976) and hyperthermophile (Koch etc., Arch.Microbiol, 155:572-578,1991; Schumann etc., FEBS Letters, 282:122-126,1991), as thermophilic sporeformer (Bacillusstearothermophilus) (Bramm etc., No. 5612202 United States Patent (USP), March 18 1997 applying date), good hot anaerobic bacillus(cillus anaerobicus) (Thermoanaerobacter finii) and the thermophilic bacterioide of acetate ethyl (Thermobacteroides acetoethylicus) (Antranikian etc., No. 4929557 United States Patent (USP), the May 29 nineteen ninety applying date), hot sulfurization hydrogen clostridium (Clostridiumthermohydrosulfuricum) (Zeikus etc., pct international patent application WO 8601831, December 9 1986 applying date), fireball bacterium (Pyrococcus furiosus) (Laderman etc., No. 5578479 United States Patent (USP), November 26 1996 applying date; Kelly etc., No. 6355467 United States Patent (USP), March 12 2002 applying date).Microorganisms α-Dian Fenmei kind is many, and the α-Dian Fenmei of different sources has many-sided different properties, thereby causes the range of application that differs from one another.Thereby need develop the new features α-Dian Fenmei continuously to satisfy industrial needs better.
About alpha-amylase gene patent and bibliographical information are arranged.Reported the alpha-amylase gene (No. 6391595 United States Patent (USP), May 21 2002 year applying date) of sulfolobus solfataricus (Sulfolobus sp.) as Kato etc.; Ito etc. have reported the alpha-amylase gene (No. 6087147 United States Patent (USP), July 11 2000 applying date) of pseudomonas (Pseudomonas sp.); Kidd etc. have reported the alpha-amylase gene (J Appl Microbiol, 92:289-96,2002) of Aeromonas hydrophila (Aeromonas hydrophila.); Kim etc. have reported the alpha-amylase gene (Appl Environ Microbiol, 65:1644-51,1999) of the hot bacterium that dwells (Thermus sp.); Dautor etc. have reported the alpha-amylase gene (Biochemistry, 38 (26), 8385-8392,1999) of bacstearothermophilus (Bacillus stearothermophilus).
At present show maximum in the acid pH scope and stable enzyme is cut activity in neutrality, but in the basic solution of pH 9-10, seldom play a role in most of α-Dian Fenmei that occurring in nature is found.Known have only the alkali alpha amylase of minority to show maximum activity in the pH of alkalescence scope.Alkali alpha amylase is meant that the suitableeest working pH of enzyme is higher than 8 α-Dian Fenmei, these alkali alpha amylases comprise a kind of enzyme (Horikoshi that is produced by bacillus (Bacillus sp.) A-40-2, K. etc., Agric.Biol.Chem., 35,1782,1971), a kind of enzyme (Boyer by bacillus NRRL B-3881 generation, E. etc., J.Bacteriol., 110,992,1972), by a kind of enzyme (Japanese publication 61-209528) of streptomycete (Streptomyces) KSM-9 generation, by a kind of enzyme (Japanese publication 62-208278) of bacillus H-167 generation, by a kind of enzyme (Japanese publication 2-49584) of bacillus A3-8 generation, and by having a liking for a kind of enzyme (Japanese publication 4-211369) that saline and alkaline coccus (Natronococcus) AH-36 produces.Above-mentioned alkali alpha amylase major part is the so-called saccharification α-Dian Fenmei that starch or starch associated sugars is decomposed into glucose, maltose or trisaccharide maltose.Though they can be effectively used in the sugar-refining industry, if their enzymes as washing composition can be had problems.Therefore, still need to seek the surfactant that is used for washing composition is had resistance and with a kind of mode starch-splitting of height random or the so-called liquefaction type alkali alpha amylase of the relevant polysaccharide of starch.
Summary of the invention
An object of the present invention is to provide a kind of gene, the α-Dian Fenmei of its a kind of thermo philic alkali of encoding.
Another object of the present invention provides a kind of thermophilic alkali alpha amylase, its can be under comparatively high temps and higher pH value condition hydrolyzed starch.
Another object of the present invention provides a kind of recombinant plasmid that can express thermophilic alkali alpha amylase.
A further object of the present invention provides a kind of reorganization bacterium that can produce thermophilic alkali alpha amylase.
To achieve these goals, the present invention adopts following technical scheme:
The present invention proposes a kind of gene of the thermophilic alkali alpha amylase of encoding, its have be selected from following a), b) or nucleotide sequence c):
A) nucleotide sequence shown in the SEQ ID NO:1;
B) because the degeneracy of genetic code is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
C) under stringent hybridization condition with above-mentioned a) or b) in sequence hybridization, and coding has the nucleotide sequence of active thermophilic alkali alpha amylase.
In one embodiment of the invention, the gene of above-mentioned coding thermophilic alkali alpha amylase has the nucleotide sequence shown in the SEQ ID NO:1.
The present invention proposes a kind of thermophilic alkali alpha amylase, its have be selected from following d), e) or aminoacid sequence f):
D) above-mentioned a), b) or c) described nucleotide sequence coded aminoacid sequence;
E) aminoacid sequence shown in the SEQ ID NO:2;
F) disappearance above-mentioned e), replace or insert the aminoacid sequence behind one or more amino acid, and the protein with this sequence there is the activity of thermophilic alkali alpha amylase.
In one embodiment of the invention, above-mentioned thermophilic alkali alpha amylase has the aminoacid sequence shown in the SEQ ID NO:2.
The present invention proposes a kind of recombinant plasmid of expressing thermophilic alkali alpha amylase, and it comprises the gene of above-mentioned coding thermophilic alkali alpha amylase at least.
In one embodiment of the invention, the carrier of above-mentioned recombinant plasmid is pET-28a (+).
The present invention proposes a kind of reorganization bacterium that produces thermophilic alkali alpha amylase, has imported the gene of above-mentioned coding thermophilic alkali alpha amylase in this bacterium.
In one embodiment of the invention, above-mentioned reorganization bacterium is intestinal bacteria for example, is preferably e. coli strain bl21.
Should be pointed out that the above-mentioned term of mentioning " stringent condition " implication in this manual is meant has formed so-called specific hybridization and has not formed non-specific hybridization under this condition.For example, this stringent condition can be, homology each other is not less than can hybridize between 70% the DNA and be lower than between the DNA of above-mentioned numerical value and can not hybridize, and preferably homology is no less than between 90% the DNA and can hybridizes.For common wash conditions in the Southern hybridization, can for example be following hybridization conditions: with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.0,7%SDS) in, 50 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.0,7%SDS, isotope-labeled nucleotide fragments), 50 ℃ of hybridization 12hr; Abandon hybridization solution, add film washing liquid I (2 * SSC and 0.1%SDS), wash film 2 times for 50 ℃, each 30min; Add film washing liquid II (0.5 * SSC and 0.1%SDS), wash film 30min for 50 ℃.
The aminoacid sequence of the thermophilic alkali alpha amylase genes encoding that the present invention relates to is compared with the aminoacid sequence of other α-Dian Fenmei of having reported, and similarity is less than 40%.
The person of ordinary skill in the field will be appreciated that, the dna sequence dna of coding thermophilic alkali alpha amylase of the present invention comprises that also coding carries out the proteinic nucleotide sequence that one or more amino acid are replaced, inserted or disappearance also still has this enzymic activity to the aminoacid sequence of the expressed enzyme molecule of nucleotide sequence shown in the SEQ ID NO:1.
In addition, the amino acid of the expressed enzyme molecule of thermophilic alkali alpha amylase gene of the present invention being carried out one or more amino acid replaces, inserts or lack resulting protein and also can reach purpose of the present invention.Thereby the present invention also comprises with the aminoacid sequence shown in the SEQ ID NO:2 having at least 70% homology, preferably has at least 90% homology, but has the active protein of thermophilic alkali alpha amylase simultaneously.The term that uses above " a plurality of " can be the number less than 100, is preferably the number less than 10.
The thermostability height of thermophilic alkali alpha amylase of the present invention, alkaline-resisting, be suitable for starch processing and other related industries, as: use in chemical industry, weaving, food, the medicine industry.
Description of drawings
Fig. 1 is the structure mode chart of the thermophilic alkali alpha amylase gene recombination plasmid pETAMY in the embodiment of the invention.
Fig. 2 is the temperature of reaction graphic representation of reorganization thermophilic alkali alpha amylase of the present invention.
Fig. 3 is the reaction pH graphic representation of reorganization thermophilic alkali alpha amylase of the present invention.
Embodiment
The present invention will be described in more detail below by specific embodiment and with reference to accompanying drawing.The embodiment that it should be understood that the following stated is only used for explanation rather than restriction the present invention.
Embodiment one
1. the extraction of the total DNA of thermophilic denitrifying bacillus NG80-2 (CGMCC No.1228)
Studies have shown that the α-Dian Fenmei that thermophilic denitrifying bacillus (Geobacillus thermodenitrificans) NG80-2 produces is particularly suitable for hydrolyzed starch polysaccharide molecule under high temperature and alkaline condition.Therefore, in the present embodiment, (this bacterial strain has been deposited in China Microbial Culture Preservation Commission common micro-organisms center to the thermophilic denitrifying bacillus NG80-2 that employing obtains from Chinese Tianjin Dagang Oilfield official 69-8 block oil-well strata water sepn, its preserving number is CGMCC No.1228), get the fresh culture bacterium liquid 3ml of its incubated overnight, centrifugal collection thalline, thalline is suspended from the 250 microlitre 50mM Tris damping fluids (pH8.0), add 10 microlitre 0.4M EDTA (pH8.0), 37 ℃ of insulation 20min behind the mixing, add 30 microlitre 20mg/ml N,O-Diacetylmuramidases afterwards, 37 ℃ are incubated 20min again behind the mixing, add 5 microlitre 20mg/ml Proteinase Ks again, behind the gentle mixing, add 20 microlitre 10%SDS again, 50 ℃ are incubated to solution and clarify, use equal-volume phenol respectively: chloroform: twice of primary isoamyl alcohol extracting, chloroform: the primary isoamyl alcohol extracting once, last supernatant solution adds the dehydrated alcohol of 2.5 times of volume precoolings, reclaim DNA, with 70% ethanol rinsing, precipitation is dissolved in 100 microlitre TE damping fluid (pH8.0,10mMTris, 1mMEDTA), add 10mg/ml RNase 2 microlitres, 65 ℃ of insulation 30min, use phenol respectively: chloroform: primary isoamyl alcohol, chloroform: each extracting of primary isoamyl alcohol once, supernatant liquor adds the dehydrated alcohol of 2.5 times of volume precoolings, reclaims DNA, washes with 70% ethanol, vacuum-drying, precipitation is dissolved in 50 microlitre TE damping fluids.The ultraviolet spectrophotometer measurement result of dna solution is A260/A280=1.95, A260=0.73.
2. the clone of thermophilic alkali alpha amylase gene and screening
Get foregoing total dna solution 0.5 microlitre (about 10ng) as template, as primer, and carry out 20 cycle P CR by the PCR loop parameter of following setting with following oligonucleotide sequence.
The PCR loop parameter of setting is as follows:
95℃，3min；95℃，30s；50℃，45s；72℃，2min；72℃，5min；4℃，2hr。
Primer sequence is as follows:
Upstream primer: 5 '-ACGAGAATTCATGGGGAACCGGCTTTTTATGC-3 ';
Downstream primer: 5 '-TGCACTCGAGTTATTCATTTGTCCGTTTTGTTC-3 ',
Above-mentioned PCR product EcoRI and XhoI double digestion, the agarose gel electrophoresis through 0.8% is cut glue recovery 1.5kb enzyme and is cut the product segment.With through same restricted type restriction endonuclease enzymolysis and cut the plasmid pET-28a (+) that glue reclaims and be connected, behind the transformed competence colibacillus bacillus coli DH 5 alpha, be applied to and contain 50 μ g/ml Kan (kalamycin), on the LB solid medium of 0.5% starch.Cultivated 12 hours for 37 ℃, cultivated 1-5 hour at 60 ℃ again, periphery of bacterial colonies has the obvious transparent circle to be positive colony, and the picking mono-clonal extracts plasmid and identifies that pET-28a (+) plasmid that is inserted with the dna sequence dna of SEQ ID NO:1 is a recombinant plasmid pETAMY (see figure 1).
(this bacterial strain can be ordered to the thick general biotechnology in development area, Tianjin development corporation, Ltd. with the recombinant plasmid pETAMY transformed competence colibacillus e. coli bl21 after identifying, article No. is 69387-3) after, be applied on the LB solid medium that contains 50 μ g/ml Kan (kalamycin).Cultivated 16～18 hours for 37 ℃, the picking mono-clonal identifies that pET-28a (+) plasmid that is inserted with the dna sequence dna of SEQ ID NO:1 is a recombinant plasmid pETAMY (see figure 1), and the recombinant escherichia coli strain that contains this plasmid is e. coli bl21 AMY.Adopt the Sanger dideoxy method that this dna fragmentation is checked order.Sequencing result shows that this gene DNA fragment total length 1536bp is begun by the ATG initiation codon, to the ending of TAA terminator codon.Protein of forming by 511 amino acid of this complete ORF coding.Belong to α-Dian Fenmei (alpha-amylase) family, the highest α-Dian Fenmei (40%) similar in appearance to thermoactinomyces vulgaris (Thermoactinomyces vulgaris).
Embodiment twoThe purifying of recombinant alpha-amylases and characteristic
Above-mentioned reorganization bacterium E.Coli BL21AMY mono-clonal is inserted 20ml to be contained in the LB substratum of 50 μ g/ml Kan, 37 ℃, 180rpm/min cultivated 12 hours, then culture is inserted the LB substratum (totally 10 are shaken bottle) that 200ml contains 50 μ g/ml Kan by 1% (V/V) inoculum size, 37 ℃, it is 0.6 o'clock that 220rpm/min cultivates A600, add IPTG to final concentration be 0.2mM, 45 ℃, 150rpm/min induced 4 hours.Centrifugal collection thalline is suspended from 20mM sodium phosphate (pH8.0) damping fluid, utilizes the ultrasonic disruption cell, and centrifuged supernatant is the crude extract of recombinant alpha-amylases.This supernatant liquor is through chelating sepharose (Chelating Sepharose) nickel affinity column chromatography purification, and the zymin that obtains shows a band on SDS-PAGE.Utilize the standard method of known protein materialization to measure the fundamental characteristics of this recombinant alpha-amylases.The molecular weight of the recombinase that records with SDS-PAGE is 60000 dalton, and is similar to the molecular weight of calculating in theory (59894 dalton); The iso-electric point pI of the recombinase that isoelectric point precipitation records is 5.828.
Embodiment threeThe determination of activity of reorganization noise thermokalite α-Dian Fenmeishuixie starch
The following reaction system of 4ml is housed: 0.1% starch solution 1ml, 20mMNa in the 20ml spiral cover test tube
2HPO
4(pH8.0), the purifying enzyme preparation 1mg of the reorganization thermophilic alkali alpha amylase that makes in the foregoing description two.60 ℃ of reaction 10min.Add 1ml 0.1M H
2SO
4Termination reaction.Get 2ml total reaction liquid and add 0.5ml 0.5%KI-I
2Solution, mixing is measured the uv-spectrophotometric value with absorption photometry under 620nm.
(1) the suitableeest enzyme temperature alive
Under the standard reaction condition, in 25-80 ℃ of scope, measure the activity (as shown in Figure 2) of above-mentioned reorganization thermophilic alkali alpha amylase, show that the optimum temperuture that keeps this enzymic activity is 60 ℃.
(2) optimum pH
Under the standard reaction condition (60 ℃), measure the activity (as shown in Figure 3) of above-mentioned reorganization thermophilic alkali alpha amylase under the condition of the different damping fluids of the 20mM in table 1, the result shows that the α-Dian Fenmei optimal pH of expressing among the recombinant bacterial strain E.Coli BL21AMY that makes up in the foregoing description one is 10.
Used damping fluid in the enzyme activity determination of table 1. thermophilic alkali alpha amylase.
| The pH scope | Damping fluid 1 | Damping fluid 2 |
| pH2-8 | 0.1M Na 2HPO 4(pH9.0) | 0.1M citric acid (pH2.0) |
| pH9-11 | 0.1M Na 2CO 3(pH11.8) | 0.1M NaHCO 3(pH8.4) |
(3) pH stability
Purifying protein is mixed with the damping fluid of pH2.0-12.0, place 30min for 25 ℃, measure enzyme activity, the result shows that enzyme work is more stable in the scope of pH7-11.5.
Experimental results show that through above-mentioned, the reorganization bacterium E.Coli BL21AMY that makes up in the foregoing description 2 expresses the alpha-amylase activity under the high-temperature alkaline condition, the reorganization thermophilic alkali alpha amylase hydrolyzable starch of its generation produces glucose and maltose, optimum temperuture is about 60 ℃, optimum pH is about 10, and enzyme work is more stable in the scope of pH7-11.5.
Though the present invention discloses as above with preferred embodiment; right its is not in order to qualification the present invention, any person of ordinary skill in the field, without departing from the spirit and scope of the present invention; can do a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
Sequence table
＜110〉Nankai University
＜120〉a kind of thermophilic alkali alpha amylase and encoding gene thereof
<130>5P13001-CN-A
<160>2
<170>PatentIn version 3.2
<210>1
<211>1536
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＜213〉thermophilic denitrifying bacillus (Geobacillus thermodenitrificans) NG80-2
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atggggaacc ggctttttat gctgttcatc cttccgttcc ttctttttta tgccatgccg 60
gttgcggcgg cggaaaaaga agaacggacg tggcaagacg aagcgattta ttttattatg 120
gtcgatcgtt ttaacaacat ggattcagcg aacgaccaag acgtcaatgt caatgatcca 180
aaagggtatt ttggcggtga cttaaaaggg gtgacagcga agctcgatta tattaaagaa 240
atgggcttca ctgccatttg gctgactccc atttttaaaa acaggccggg cggctatcat 300
ggctattgga tcgaggactt ttacgaagtc gacccgcatt ttggcacgct tgatgacctc 360
aagacgctcg tcaaagaggc gcataagcgc gatatgaaag tgattttgga tttcgtggct 420
aaccatgtcg gctacgacca tccgtggctt catgacccgg cgaaaaaaga ttggttccat 480
ccgaagaaag agattttcga ctggaacagc caagagcagg tggaaaacgg ttgggtgtat 540
ggacttcccg atttggcgca ggaaaatccg gaagtaaaaa actatttaat tgacgcagcc 600
aaatggtgga ttaaagaaac cgacattgac ggttaccgac tcgacatggt acgccatgtg 660
ccgaaatcgt tctggcaaga gtttgcgaaa gaagtgaaag cggtaaaaaa agactttttt 720
ctcctcggcg aagtgtggag tgatgatccg cgctatatcg ctgattacgg aaagtatggc 780
atcgacgggt ttgtcgatta cccgctgtac ggtgcggtga agcagtctct tgccaaacgt 840
gatgcatcgc ttcggccgct ctatgatgta tgggagtata acaagacatt ttacgaccgc 900
ccgtatttgc ttggatcatt tttggacaac catgataacg tccgatttac gaaactcgtc 960
attgatcatc ggaacaatcc gatttcacgc atgaaagtag cgatgacgta tttgttcact 1020
gcgccgggca ttccgattat gtactacgga acagaaattg cgatgactgg aggtcctgat 1080
ccggacaatc gccgcctgat ggacttccgt gccgatccag aaatcatcga ttatttaaag 1140
aaagtgggcc cgctccgcca acagttgccg tcattgcggc gcggcgattt tacgttgttg 1200
tatgaacaag acggcatggc ggtgtttaaa cgccaataca aagacgaaac gacggtcatt 1260
gccattaaca acacgagtga aacgaagcac gttcatctga cgaacgaaca gctgccgaaa 1320
aacaaggaat tgcgtggctt tttgctcgat gacctcgtcc gtggcgatga ggatggctat 1380
gatattgtct tggatcgtga aacagcagaa gtgtacaaat tacggaacaa aacgggagtt 1440
aacgtgccgt ttatcgttgc aatggttgcg gtttacgcac tatttatctt gtttttatac 1500
atggtgaaaa aacgaacaaa acggacaaat gaataa 1536
<210>2
<211>511
<212>PRT
＜213〉thermophilic denitrifying bacillus (Geobacillus thermodenitrificans) NG80-2
<400>2
Met Gly Asn Arg Leu Phe Met Leu Phe Ile Leu Pro Phe Leu Leu Phe
1 5 10 15
Tyr Ala Met Pro Val Ala Ala Ala Glu Lys Glu Glu Arg Thr Trp Gln
20 25 30
Asp Glu Ala Ile Tyr Phe Ile Met Val Asp Arg Phe Asn Asn Met Asp
35 40 45
Ser Thr Asn Asp Gln Asp Val Asn Val Asn Asp Pro Lys Gly Tyr Phe
50 55 60
Gly Gly Asp Leu Lys Gly Val Thr Ala Lys Leu Asp Tyr Ile Lys Glu
65 70 75 80
Met Gly Phe Thr Ala Ile Trp Leu Thr Pro Ile Phe Lys Asn Arg Pro
85 90 95
Gly Gly Tyr His Gly Tyr Trp Ile Glu Asp Phe Tyr Glu Val Asp Pro
100 105 110
His Phe Gly Thr Leu Asp Asp Leu Lys Thr Leu Val Lys Glu Ala His
115 120 125
Lys Arg Asp Met Lys Val Ile Leu Asp Phe Val Ala Asn His Val Gly
130 135 140
Tyr Asp His Pro Trp Leu His Asp Pro Ala Lys Lys Asp Trp Phe His
145 150 155 160
Pro Lys Lys Glu Ile Phe Asp Trp Asn Ser Gln Glu Gln Val Glu Asn
165 170 175
Gly Trp Val Tyr Gly Leu Pro Asp Leu Ala Gln Glu Asn Pro Glu Val
180 185 190
Lys Asn Tyr Leu Ile Asp Ala Ala Lys Trp Trp Ile Lys Glu Thr Asp
195 200 205
Ile Asp Gly Tyr Arg Leu Asp Met Val Arg His Val Pro Lys Ser Phe
2l0 215 220
Trp Gln Glu Phe Ala Lys Glu Val Lys Ala Val Lys Lys Asp Phe Phe
225 230 235 240
Leu Leu Gly Glu Val Trp Ser Asp Asp Pro Arg Tyr Ile Ala Asp Tyr
245 250 255
Gly Lys Tyr Gly Ile Asp Gly Phe Val Asp Tyr Pro Leu Tyr Gly Ala
260 265 270
Val Lys Gln Ser Leu Ala Lys Arg Asp Ala Ser Leu Arg Pro Leu Tyr
275 280 285
Asp Val Trp Glu Tyr Asn Lys Thr Phe Tyr Asp Arg Pro Tyr Leu Leu
290 295 300
G1y Ser Phe Leu Asp Asn His Asp Asn Val Arg Phe Thr Lys Leu Val
305 310 315 320
Ile Asp His Arg Asn Asn Pro Ile Ser Arg Met Lys Val Ala Met Thr
325 330 335
Tyr Leu Phe Thr Ala Pro Gly Ile Pro Ile Met Tyr Tyr Gly Thr Glu
340 345 350
Ile Ala Met Thr Gly Gly Pro Asp Pro Asp Asn Arg Arg Leu Met Asp
355 360 365
Phe Arg Ala Asp Pro Glu Ile Ile Asp Tyr Leu Lys Lys Val Gly Pro
370 375 380
Leu Arg Gln Gln Leu Pro Ser Leu Arg Arg Gly Asp Phe Thr Leu Leu
385 390 395 400
Tyr Glu Gln Asp Gly Met Ala Val Phe Lys Arg Gln Tyr Lys Asp Glu
405 410 415
Thr Thr Val Ile Ala Ile Asn Asn Thr Ser Glu Thr Lys His Val His
420 425 430
Leu Thr Asn Glu Gln Leu Pro Lys Asn Lys Glu Leu Arg Gly Phe Leu
435 440 445
Leu Asp Asp Leu Val Arg Gly Asp Glu Asp Gly Tyr Asp Ile Val Leu
450 455 460
Asp Arg Glu Thr Ala Glu Val Tyr Lys Leu Arg Asn Lys Thr Gly Val
465 470 475 480
Asn Val Pro Phe Ile Val Ala Met Val Ala Val Tyr Ala Leu Phe Ile
485 490 495
Leu Phe Leu Tyr Met Val Lys Lys Arg Thr Lys Arg Thr Asn Glu
500 505 510
Claims (9)
1. the gene of the thermophilic alkali alpha amylase of encoding, this gene have be selected from following a), b) or nucleotide sequence c):
A) nucleotide sequence shown in the SEQ ID NO:1;
B) because the degeneracy of genetic code is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
C) under stringent hybridization condition with above-mentioned a) or b) in sequence hybridization, and coding has the nucleotide sequence of active thermophilic alkali alpha amylase.
2. the gene of coding thermophilic alkali alpha amylase according to claim 1 is characterized in that this gene has the nucleotide sequence shown in the SEQ ID NO:1.
3. thermophilic alkali alpha amylase, this enzyme have be selected from following d), e) or aminoacid sequence f):
D) the described nucleotide sequence coded aminoacid sequence of claim 1;
E) aminoacid sequence shown in the SEQ ID NO:2;
F) disappearance above-mentioned e), replace or insert the aminoacid sequence behind one or more amino acid, and the protein with this sequence there is the activity of thermophilic alkali alpha amylase.
4. thermophilic alkali alpha amylase according to claim 3 is characterized in that this enzyme has the aminoacid sequence shown in the SEQID NO:2.
5. recombinant plasmid of expressing thermophilic alkali alpha amylase, this plasmid comprises claim 1 or 2 described genes at least.
6. recombinant plasmid according to claim 5, the carrier that it is characterized in that this recombinant plasmid is pET-28a (+).
7. a reorganization bacterium that produces thermophilic alkali alpha amylase has imported claim 1 or 2 described genes in this reorganization bacterium.
8. reorganization bacterium according to claim 7 is characterized in that this reorganization bacterium is intestinal bacteria.
9. reorganization bacterium according to claim 8 is characterized in that above-mentioned intestinal bacteria are e. coli strain bl21.
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| CN111808784A (en) * | 2020-08-04 | 2020-10-23 | 中国海洋大学 | Heat-denitrifying Geobacillus Y62 for producing high-temperature-resistant alpha-amylase and application thereof |
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| US4628031A (en) * | 1984-09-18 | 1986-12-09 | Michigan Biotechnology Institute | Thermostable starch converting enzymes |
| BR9006818A (en) * | 1989-06-29 | 1991-08-06 | Gist Brocades Nv | MUTANT MICROBIAL AMYLASES WITH GREATER THERMAL, ACID AND / OR ALKALINE STABILITY |
| JPH04211369A (en) * | 1990-02-01 | 1992-08-03 | Nippon Shokubai Co Ltd | Halophilic alkali amylase and production thereof |
| NZ250048A (en) * | 1992-10-28 | 1994-10-26 | Enzyme Bio Systems Ltd | Production of maltodextrins by selective hydrolysation of starches by enzymatic methods |
| JP4426716B2 (en) * | 2000-10-11 | 2010-03-03 | 花王株式会社 | High productivity α-amylase |
| CN1313608C (en) * | 2004-01-18 | 2007-05-02 | 中国科学院微生物研究所 | Basic alpha-diastase, and its coding gene and production method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808784A (en) * | 2020-08-04 | 2020-10-23 | 中国海洋大学 | Heat-denitrifying Geobacillus Y62 for producing high-temperature-resistant alpha-amylase and application thereof |
| CN111808784B (en) * | 2020-08-04 | 2021-06-08 | 中国海洋大学 | Heat-denitrifying Geobacillus Y62 for producing high-temperature-resistant alpha-amylase and application thereof |
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