CN104178471A - Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase - Google Patents
Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase Download PDFInfo
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- CN104178471A CN104178471A CN201410261362.6A CN201410261362A CN104178471A CN 104178471 A CN104178471 A CN 104178471A CN 201410261362 A CN201410261362 A CN 201410261362A CN 104178471 A CN104178471 A CN 104178471A
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Abstract
The invention provides low-temperature alpha-amylase originated from a fungus. The low-temperature alpha-amylase originated from the fungus is a protein of (a) or (b): (a) a protein which is formed by the following amino acid sequences shown in SEQ ID NO.19; (b) a protein which has the function of the alpha-amylase and is derived from (a) by means of substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequences shown in the SEQ ID NO.19. The coding gene provided by the invention is deoxyribonucleotide shown in (1) or (2): (1) a nucleotide sequence shown in SEQ ID NO.17; (b) a nucleotide sequence shown in SEQ ID NO.18. A recombinant host cell provided by the invention comprises a recombinant expression vector with the above coding gene. The optimum temperature of the alpha-amylase according to the invention is 40 DEG C which is 10 DEG C lower than that of normal-temperature amylase. The alpha-amylase is good in low-temperature adaptability, still maintains the activity of over 20% of highest activity at 0-30 DEG C and has a huge application prospect in detergent and textile desizing.
Description
Technical field
The present invention relates to a kind of fungal alpha-amylase at low temperatures with good activity, the invention still further relates to encoding gene and the application of this low-temperature amylase.
Technical background
α-amylase (α-1,4-D-glucan-glucanhydrolase, EC3.2.1.1) belong to endo-type amylase, can be to amylopectin amylose starch or other-1,4-dextran molecule inside-Isosorbide-5-Nitrae-glycosidic link is hydrolyzed in random mode, generates the general name of the class of enzymes of straight chain that length do not wait and branched oligosaccharides, be starch industry Major Enzymes, in fields such as food-processing, washing composition, weaving destarch, paper industries, there is important commercial value.Low-temperature amylase (cold-adapted amylase) refers to can effectively bring into play the amylase of katalysis at low temperatures, general optimum temperuture is at 35～45 ℃, except the above application, can also be for low temperature environment biological restoration, in industrial application because its less energy-consumption has broad prospects.α-amylase is extensively present in various biologies such as comprising people, animal, plant, fungus and bacterium, since the separated α-amylase of reported first in 1956, at present separated and identify surpass 120 kinds of α-amylase, microbe-derived α-amylase accounts for major part in quantity.The genus that can produce α-amylase in microorganism has: Bacillus, Thermomonospor, Acinetobacter, Pseudomonas, Streptomyces, Aspergillus, Penicillus etc.α-amylase in industrial a large amount of uses is mainly derived from subtilis (Bacillus subtilis) at present, lichens bud pole bacterium (Bacillus licheniformis), separate starch bud pole bacterium (Bacillusamyloliquefaciens), aspergillus niger (Aspergillus niger) and aspergillus oryzae (Aspergillus oryzae).
The α-amylase of applying on Vehicles Collected from Market be mainly by aspergillus oryzae fermentative production, obtained in warm fungal amylase (TAKA-amylase, fungal amylase) and the thermophilic bacteria α-amylase that produces of genus bacillus, although enzyme activity is higher, but optimum temperuture higher (50 ℃-90 ℃), and very low at lesser temps (normal temperature) vigor, cause needing heat treated at washing composition and weaving in destarch, technique is loaded down with trivial details and increase energy consumption; And low-temperature amylase effectively overcomes the defect in this technique because of its Tough structure performance, and because its thermal lability can destroy structure under middle temperature environment, reduce and pollute.
Although filter out some new low temperature alpha-amylases both at home and abroad, produce bacterium, generally slower because of psychrophile growth, expression vigor is very low, and has limited in industrial application.Therefore, how to screen and both had advantageous property, the low temperature alpha-amylase that can obtain again high expression level vigor becomes study hotspot.As far back as 1992, Georges Feller etc. once reported that alternately Zymomonas mobilis (Alteromonas haloplanctis) can produce low temperature alpha-amylase (Georges Feller et al.1992.Purification, Characterization, and Nucleotide Sequence of the Thermolabile α-Amylase from the Antarctic Psychrotroph Alteromonas haloplanctis A23.THE JOURNAL OF BIOLOGICAL CHEMISTR.267 (8)): 5217-5221.), in recent years, a series of low-temperature amylases that come from psychrotropic bacteria are in succession out screened and in intestinal bacteria, realized heterogenous expression.Simultaneously, many South Pole is produced amylase low temperature fungi and is isolated and identified out, 1997, M.Fenice á L etc. is separated to the different G.pannorum var.pannorum that five strains all have amylase activity from Victoria island, the South Pole, wherein no.1 has demonstrated significant vigor, the application prospect (M.Fenice á L et al.1997.Production of extracellular enzymes by Antarctic fungal strains.Polar Biol, 17:275-280) that has industrialization; 2011, Abiramy Krishn etc. has screened the fungi of secreting amylase under several strain low temperature again on island, the South Pole, its main Pseudomonas is Geomyces pannorum (Abiramy Krishn et al.2011.Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica.Polar Biol, 34:1535 – 1542).Although Geomyces pannorum is had to considerable amylase activity, but there is no report about its amylase gene sequence and protein sequence, and due to this bacterium culture condition restriction, also do not have so far separation and purification to go out the amylase of this bacterium, its zymologic property is not yet studied.
Summary of the invention
The object of this invention is to provide a kind of low temperature alpha-amylase from fungi and encoding gene thereof.
Low temperature alpha-amylase from fungi provided by the invention is following (a) or protein (b): the protein (a) being comprised of the aminoacid sequence shown in SEQ ID NO.19; (b) by the amino acid residue sequence of SEQ ID NO.19 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have α-amylase function by (a) derivative protein.
This low temperature alpha-amylase has the sequence with aminoacid sequence at least 70% homology shown in SEQ ID NO.19.More preferably, this low temperature alpha-amylase has the sequence with aminoacid sequence at least 80% homology shown in SEQ ID NO.19.Further preferably, this low temperature alpha-amylase has the sequence with aminoacid sequence at least 90% homology shown in SEQ ID NO.19.Best, this low temperature alpha-amylase has the sequence with aminoacid sequence at least 99% homology shown in SEQ ID NO.19.
The encoding gene of low temperature alpha-amylase provided by the invention is selected from the deoxyribonucleotide of (1)-(4) as follows: (1) has nucleotide sequence shown in SEQ ID NO.17; (2) there is nucleotide sequence shown in SEQ ID NO.18; (3) there is the sequence with nucleotide sequence at least 70% homology shown in described (1) or (2); (4) this nucleotide sequence is not limited to sequence shown in SEQ ID NO.17 or SEQ ID NO.18, obtains protein that as shown in SEQ ID NO.19 aminoacid sequence forms or the nucleotide sequence of its derivative functionally similar protein and all fall in this patent protection domain after any codon optimization.
This encoding gene has the sequence with nucleotide sequence at least 70% homology shown in nucleotide sequence shown in SEQ ID NO.17 or SEQ ID NO.18.More preferably, this encoding gene has the sequence with nucleotide sequence at least 80% homology shown in nucleotide sequence shown in SEQ ID NO.17 or SEQ ID NO.18.Further preferably, this encoding gene has the sequence with nucleotide sequence at least 90% homology shown in nucleotide sequence shown in SEQ ID NO.17 or SEQ ID NO.18.Best, this encoding gene has the sequence with nucleotide sequence at least 99% homology shown in nucleotide sequence shown in SEQ ID NO.17 or SEQ ID NO.18.
Recombinant expression vector provided by the invention comprises above-mentioned encoding gene.
Described recombinant expression vector is for inserting the recombinant expression vector that above-mentioned encoding gene obtains between the multiple clone site at aspergillus expression vector pBC12FNH.
Recombinant host cell provided by the invention comprises above-mentioned recombinant expression vector.
Described recombinant host cell is that above-mentioned recombinant expression vector is transferred to the recombinant host cell obtaining in aspergillus oryzae RIB40pyrF auxotrophic strain PF1 (CGMCC NO:9129).
The invention provides a kind of method of preparing recombinant fungus low temperature alpha-amylase.
The method of preparing recombinant fungus low temperature alpha-amylase provided by the present invention, is the above-mentioned recombinant bacterium that contains low temperature alpha-amylase encoding gene of low temperature (20 ℃) fermentation culture, obtains low temperature alpha-amylase.
The present invention successfully discloses Geomyces Pannorum alpha-amylase gene and protein amino acid sequence.According to 40 ℃ of low temperature alpha-amylase optimum temperutures of the present invention, and under low temperature, enzyme is alive higher, is suitable for the requirement of industrial application.The recombinant host cell that the present invention obtains, occurs at low temperatures ferment is cultivated, and is suitable for the expression of above-mentioned low temperature alpha-amylase gene.
Accompanying drawing explanation
Fig. 1 shows expression vector pBC12FNHA2 design of graphics;
Fig. 2 shows GpA2 at the protein electrophoresis figure of aspergillus oryzae RIB40-PF1-A2 recombinant bacterium expression and purity;
It is 40 ℃ (under the condition of pH5.0, measuring) that Fig. 3 shows GpA2 optimum temperature, and keeps high enzyme vigor at 0～40 ℃;
It is stable under 40 ℃ of conditions that Fig. 4 shows GpA2, under 40 ℃ of conditions, hatches 30min, still has 65% residual enzyme activity, and at 60 ℃, hatch the basic inactivation of 5min; Wherein 40 ℃ (■), 50 ℃ (●), 60 ℃ (▲);
It is 5.0 that Fig. 5 shows the suitableeest action pH of GpA2, and in pH3.0～6.0 are interval, (under the condition of 40 ℃, measuring) has high enzyme vigor;
Fig. 6 show GpA2 in the interval of pH4.0～9.0 30 ℃ all there is good pH stability after hatching 30min.
Embodiment
Following examples are the non-limiting the scope of protection of the invention for the present invention is described only.
Test method described in following embodiment, if no special instructions, is ordinary method; Described reagent or consumptive material, if no special instructions, all can obtain by commercial sources.As < < molecular cloning: laboratory manual > > (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in is carried out, or the condition of advising according to manufacturer.Damping fluid and the substratum in embodiment, used are as follows:
(1)TE(1000ml)：
ZnSO
47H
2o0.35g, MnCl
24H
2o0.5g, H
3bO
30.03g, CoCl
26H
2o0.945g, CuCl
22H2O0.01g, NiCl6H
2o0.12g, NaMoO
42H
2o0.18g, 6M HCl13ml, adds deionized water and is settled to 500mL.
(2) sodium phosphate buffer (100ml, pH5.8):
Na
2HPO
4·12H
2O0.5788g，NaH
2PO
4·2H
2O2.8996g。
(3) Tris-HCl damping fluid (100ml, pH7.5):
Tris0.6057g, regulates pH to 7.5 with HCl.
(4) solution I:
NaCl0.7M, sucrose 1.0g, is dissolved in 50ml sodium phosphate buffer, and constant volume (adding water) is to 100ml.
(5) solution II:
Sorbyl alcohol 22.3085g, CaCl
20.578g, NaCl0.2045g, is dissolved in 20ml Tri-HCl damping fluid, and constant volume (adding water) is to 100ml.
(6) solution III:
PEG400030g, CaCl
20.289g, is dissolved in 10ml Tri-HCl damping fluid, and constant volume (adding water) is to 50ml.
(7) CM substratum (100ml):
NaNO
30.6g, KCl0.05g, KH
2pO
40.08g, K
2hPO
40.104g, glucose 1g, MgSO
40.052g, Peptone0.2g, Yeast extract0.1g, uridine 0.1221g, TE100ul; If be made into inclined-plane, add 1.5～2.0g agar powder.
(8) MM substratum (100ml):
NaNO
30.6g, KCl0.05g, KH
2pO
40.08g, K
2hPO
40.104g, glucose 1g, MgSO
40.052g, TE100ul, sucrose 20.54g.
Agar powder: upper strata 0.8g, the 1.2g of lower floor.
(9) fermention medium (100ml):
Dextrin 2g, sucrose 0.3g, Peptone0.1g, Yeast extract0.5g, NaNO
30.1g, MgSO
47H
2o0.05g, FeSO
47H
2o0.001g.
The amplification of embodiment 1. low temperature alpha-amylase genes
1.1 bacterial strains and cultivation thereof
From biological scienology institute of Malaysian university problem collaborative project, receive Geomyces pannorum AK07KGI1001R1-2 (1) (strain identification NCBI accession number: JF720026.biramy Krishn et al.2011.Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica.Polar Biol, 34:1535 – 1542).
With sterilized water, from the PDA inclined-plane of cultivating 10 days, wash lower G.pannorum spore, inoculation liquid nutrient medium, cultivates for 20 ℃ and collects mycelium in 7 days, for genome, extracts; With toothpick inoculation starch solids substratum, cultivate for 20 ℃ and within 5 days, collect mycelium, for total RNA, extract.
1.2 genomes extract
With reference to Omega fungal gene group, extract test kit specification sheets
1.3 total RNA extract
According to Takara RNA, extracting reagent specification sheets operates.
1.4cDNA the first chain is synthetic
The total mRNA of extracting of take is template, by the method for Takara test kit, obtains cDNA the first chain, and PCR system and step are as follows:
65 ℃ of insulation 5min, cooling rapidly on ice, in above-mentioned reaction tubes, add
42 ℃ of 45min, 95 ℃ of 5min, cooled on ice.
1.5Geomyces pannorum α-amylase conservative region clone
On NCBI, find and the bacterial classification of the close kind of Geomyces pannorum and the α-amylase aminoacid sequence of other filamentous fungus.At the Position Design forward degenerate primer JGpf1 of α-amylase amino acid conservative region TDRFARTDGS, at the Position Design reverse takeover primer JGpr1 of amino acid conservative region RGMYLMVD.Wherein:
The sequence of JGpf1 is SEQ ID NO.1:ACNGA TMGTN TTGCR MGBAC (this primer is synthesized by bio-engineering corporation of Shanghai Jierui Biology Engineering Co., Ltd, lower same)
The sequence of JGpr1 is SEQ ID NO.2:ACNAY RTCRA CCATN ARRTA CAT
The Geomyces pannorum genome of take is template, with degenerate primer JGpf1 and JGpr1, carries out pcr amplification α-amylase conservative region, and PCR system is:
Reaction conditions is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 25s, 30 circulations; 72 ℃ are extended 10min; Be kept at 4 ℃.PCR product is carried out to agarose gel electrophoresis, reclaim, connect pMD19-TSimple Vector, bacterium colony PCR checking positive colony send order-checking, and order-checking is completed by Hua Da gene.
Because the amylase fragment obtaining is only about 400bp, sequence is less, based on DNA fragmentation obtained above, on NCBI, carry out Blast x, the higher α-amylase sequence of having reported of the homology degree of take is as according to again designing degenerate primer JSGpf2/JTGpr2, obtain longer target α-amylase sequence, amino acid conservative region is respectively IDGLRVD and ENHDVARF.Wherein
The sequence of JSGpf2 is SEQ ID NO.3:ATYGAYGGBYTBMGNGTTGA
The sequence of JTGpr2 is SEQ ID NO.4:RAADCGNGSYWSGTCRTGRTTBTC
Its subsequent process as mentioned above.
Based on two α-amylase conservative fragments obtained above, at design specific primers at both ends Gpaf/Gpar, wherein
The sequence of Gpaf is SEQ ID NO.5:GTTTGCGAGCACGGACGGC
The sequence of Gpar is SEQ ID NO.6:AGGTCGTGGTTTTCGAGGAAGTTCG
The sequence of pcr amplification α-amylase conservative fragments.PCR reaction system is:
Reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min, 10 circulations; 72 ℃ are extended 10min; Be kept at 4 ℃.
1.6 α-amylase full length DNA clones
Based on α-amylase conservative fragments DNA sequence dna obtained above, respectively at 3 Auele Specific Primers of upstream and downstream design (5 ' end: 5-1SP1,5-1SP2,5-1SP3; 3 ' end: 3-1SP1,3-1SP2,3-1SP3).The Geomyces pannorum genome of take is template, with Auele Specific Primer and chromosome walking test kit (Genome Walking Kit, purchased from Dalian precious biotechnology company limited) four kinds of degenerate primer AP1, the AP2, AP3, the AP4 that provide carry out hot asymmetric PCR reaction, by three nest-type PRCs reactions, obtains the unknown nucleotide sequence of conservative fragments both sides.Wherein:
The sequence of 5-1SP1 is SEQ ID NO.7:CCCAGCATATTTCGGTGGAGCTTTG
The sequence of 5-1SP2 is SEQ ID NO.8:AGCCGCATTACACTGGAGAAATCCA
The sequence of 5-1SP3 is SEQ ID NO.9:CCGCAGTAGTCGGTGATCCCACAAG
The sequence of 3-1SP1 is SEQ ID NO.10:TGATACAGCCAAGCATGTCCCACTG
The sequence of 3-1SP2 is SEQ ID NO.11:GCCTTGGATGGAGAGTCTGCATACG
The sequence of 3-1SP3 is SEQ ID NO.12:CATCAAAGCCTTTATGGGAGGGAGC
Operating process is as follows:
(1) 1stPCR, reaction system:
Reaction conditions is: 94 ℃ of 1min, 98 ℃ of 1min; 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 5 circulations; 94 ℃ of 30s, 25 ℃ of 3min, 72 ℃ of 2min; 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, 15 circulations; 72 ℃ of 10min; Be kept at 4 ℃.
(2) 2ndPCR, reaction system:
Reaction conditions: 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, 15 circulations; 72 ℃ of 10min; Be kept at 4 ℃.
(3) 3rdPCR, reaction system:
Reaction conditions: 94 ℃ of 30s, 0 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, 15 circulations; 72 ℃ of 10min; Be kept at 4 ℃.
2nd and 3rd PCR reaction solution are carried out to agarose gel electrophoresis, reclaim clear band, connect pMD19-T Simple Vector, bacterium colony PCR verifies positive colony, send order-checking, and order-checking is completed by Hua Da gene.
Sequencing result is spliced, then, at design specific primers at both ends GpA2f1 and the GpA2r1 of splicing sequence, take genome as template, carry out total length PCR checking, its process as mentioned above.Wherein:
The sequence of GpA2f1 is SEQ ID NO.13:GTTTCCCCTTTGACATACCAAATCA
The sequence of GpA2r1 is SEQ ID NO.14:TTGCTTGGTAATTGTTCCTTCCTTG
1.7 α-amylase cDNA clones
Analyze the 1.6 alpha-amylase gene DNA sequence dnas that obtain, according to conserved amino acid sequence with 3 multiple recursion, simultaneously in conjunction with NCBI BlastX comparison result, the initiator codon ATG having found in possibility maximum and termination codon codon TAA.On codon, predict website
(
http:// linux1.softberry.com/berry.phtml? group=programs & subgroup=gfind & topic=fgenesh), obtained main possible intron, the Auele Specific Primer of design ORF:
The sequence of GpA2F is SEQ ID NO.15:ATGCATCTACCTTTGTTTCAGATAA
The sequence of GpA2R is SEQ ID NO.16:TTAAGCACATAAACTGCCCTTGTTC
The Geomyces pannorum cDNA of take is template, with Auele Specific Primer GpA2F and GpA2R, carries out pcr amplification α-amylase ORF, and PCR system is:
Reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1.5min, 30 circulations; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 1min, 10 circulations; 72 ℃ are extended 10min; Be kept at 4 ℃.
The genome of take carries out PCR as template simultaneously, and process is described in 1.5, and electrophoresis validating DNA is greater than cDNA.
PCR product is carried out to agarose gel electrophoresis, reclaim, connect pMD19-T Simple, bacterium colony PCR checking positive colony send order-checking, and order-checking is completed by Hua Da gene, relatively draws intron sequences.Obtain thus the complete α-amylase cDNA sequence of Geomyces pannorum, i.e. open reading frame.
Sequencing result is compared and analyzed on GenBank to be shown, the alpha-amylase gene DNA obtaining is comprised of 1671 Nucleotide, and sequence is as shown in SEQ ID NO.17:
ATGCATCTACCTTTGTTTCAGATAACCCAGGCGACGAGCCTGCTTCTCACTGCCTCCTCCTTCTTCCCTATCGCAGCCGCAAAAACCGCCGCGGAATGGCGTGAACTATCCATCTACCAGGTCCTAACTGACCGGTTTGCAACCACGGACGGCACCTCCCCAACTTGTGGGATCACCGACTACTGCGGCGGAACGTGGAAGGGGATAGAGAATAAACTTGATTACATCCAGGGAATGGGGTTTGATGCAGTTTGGATTTCTCCAGTGTAATGCGGCTTGTAACCACCCAGGTAACAGTTGACTAACACTGTAGCAGGGTCCATAACATTGATGGGGAAACTCCTGCCGGCTATGCGTATCATGGGTGTGCGTTCTTCCTTTAACCCGCACTCACTAACCATGGGGTTATTCTAACGTCCAAGACAGATTGGGCTGATAACCCATACACGCTCAACGATCACTTCGGCACTACAGATGACCTCAAGAGCCTTTCAGACGCTCTACACGGGCGCGGCATGTCACTGATGGTCGACATTGTAGTCAATCACTTTGGATCAATTCAGGATTCAAGCTCTGTAGACTACAGTGCATACCCTTCGCCGTTCAATGACGCCTCGGCGTTCCATTCGCCTTGCGCAATTGACTACAAGGACCAAGCGTCCATTGAGAATTGCTGGGTTGTGACTACACCTGCTCCAGCCCTTCCTGATGTCAATACCGAAGGCGCAGCTATCTATGGAGCTCTAGTGGACAGTGTTGTTGACCTTGTTAGCAACTATAACATCGACGGAATCCGTCTTGATACAGCCAAGCATGTCCCACTGGAAGCCCTTACTCAGTTCCAAGAGGCCATCGGTGTCTTTGTTACAGGAGAGGCCTTGGATGGAGACTCTGCATACGTCTCGGAATATCAAGGCCCACTTAATAGCGCAATCAATTATCCGCTCTGGTACCCTCTCATCAAAGCCTTTATGGGAGGGAGCTTCGATGAGCTCTCAGCAATGATTAGCACTGAAGCGACGGCCTTCTCCGACGTTAACGCACTGACGAACTTCCTCGATAACCACGACCAACCGCGCATTGCAAGTGTGGCCGGCGATGACGAGGTCAGAGACAAAAATGCGGTGACATTCCTGATGTTCACTTCTGGGATTCCTATGGTTTACTACGGCTTTGAGCAGCGATTCAGCGGCGCAGCTGACCCAGACAACCGCGAAACACTCTGGACTAGCGGTTACGACACCACCACCGCACTGTACCAACACATTGCAAAATTGCACGAGATCAGGGGCGTTGCCTCGAATGTTACTGACAAGGCGACCTATTTCTCGTCCAACGTGGCCGTCCTCGGCACTTCAACCGCATACATGGCCATAGAGCGTGGGCCTATCGTGGCAGTGGTGTCAAATGTTGGTGCGGCGGGCACAAGCGACGGTTTTGACGTTACAGGATCGAAGTTTGCTTCTGGAGACTCAGTTGCTGACCTTCTAGATTGTGCCACTACGGCGACGGTGGGTGACTCCGGCGCGTTTACAAGTCCATCGAACAATGGGGAAGCTAGAGTAAGTAATAAATTATGTTCTTTCCCCTCGGAATCGAGACCTTGAATGGCTGACTTCTCTCTCATAGATCTGGATACAGGCGGAGAACAAGGGCAGTTTATGTGCTTAA
The open reading frame cDNA of this alpha-amylase gene cDNA is comprised of 1494 Nucleotide, and sequence is as shown in SEQ ID NO.18:
ATGCATCTACCTTTGTTTCAGATAACCCAGGCGACGAGCCTGCTTCTCACTGCCTCCTCCTTCTTCCCTATCGCAGCCGCAAAAACCGCCGCGGAATGGCGTGAACTATCCATCTACCAGGTCCTAACTGACCGGTTTGCAACCACGGACGGCACCTCCCCAACTTGTGGGATCACCGACTACTGCGGCGGAACGTGGAAGGGGATAGAGAATAAACTTGATTACATCCAGGGAATGGGGTTTGATGCAGTTTGGATTTCTCCAGTGGTCCATAACATTGATGGGGAAACTCCTGCCGGCTATGCGTATCATGGGTATTGGGCTGATAACCCATACACGCTCAACGATCACTTCGGCACTACAGATGACCTCAAGAGCCTTTCAGACGCTCTACACGGGCGCGGCATGTCACTGATGGTCGACATTGTAGTCAATCACTTTGGATCAATTCAGGATTCAAGCTCTGTAGACTACAGTGCATACCCTTCGCCGTTCAATGACGCCTCGGCGTTCCATTCGCCTTGCGCAATTGACTACAAGGACCAAGCGTCCATTGAGAATTGCTGGGTTGTGACTACACCTGCTCCAGCCCTTCCTGATGTCAATACCGAAGGCGCAGCTATCTATGGAGCTCTAGTGGACAGTGTTGTTGACCTTGTTAGCAACTATAACATCGACGGAATCCGTCTTGATACAGCCAAGCATGTCCCACTGGAAGCCCTTACTCAGTTCCAAGAGGCCATCGGTGTCTTTGTTACAGGAGAGGCCTTGGATGGAGACTCTGCATACGTCTCGGAATATCAAGGCCCACTTAATAGCGCAATCAATTATCCGCTCTGGTACCCTCTCATCAAAGCCTTTATGGGAGGGAGCTTCGATGAGCTCTCAGCAATGATTAGCACTGAAGCGACGGCCTTCTCCGACGTTAACGCACTGACGAACTTCCTCGATAACCACGACCAACCGCGCATTGCAAGTGTGGCCGGCGATGACGAGGTCAGAGACAAAAATGCGGTGACATTCCTGATGTTCACTTCTGGGATTCCTATGGTTTACTACGGCTTTGAGCAGCGATTCAGCGGCGCAGCTGACCCAGACAACCGCGAAACACTCTGGACTAGCGGTTACGACACCACCACCGCACTGTACCAACACATTGCAAAATTGCACGAGATCAGGGGCGTTGCCTCGAATGTTACTGACAAGGCGACCTATTTCTCGTCCAACGTGGCCGTCCTCGGCACTTCAACCGCATACATGGCCATAGAGCGTGGGCCTATCGTGGCAGTGGTGTCAAATGTTGGTGCGGCGGGCACAAGCGACGGTTTTGACGTTACAGGATCGAAGTTTGCTTCTGGAGACTCAGTTGCTGACCTTCTAGATTGTGCCACTACGGCGACGGTGGGTGACTCCGGCGCGTTTACAAGTCCATCGAACAATGGGGAAGCTAGAATCTGGATACAGGCGGAGAACAAGGGCAGTTTATGTGCTTAA
497 amino acid of this cDNA coding, its sequence is as shown in SEQ ID NO.19:
MHLPLFQITQATSLLLTASSFFPIAAAKTAAEWRELSIYQVLTDRFATTDGTSPTCGITDYCGGTWKGIENKLDYIQGMGFDAVWISPVVHNIDGETPAGYAYHGYWADNPYTLNDHFGTTDDLKSLSDALHGRGMSLMVDIVVNHFGSIQDSSSVDYSA
YPSPFNDASAFHSPCAIDYKDQASIENCWVVTTPAPALPDVNTEGAAIYGALVDSVVDLVSNYNIDGIRLDTAKHVPLEALTQFQEAIGVFVTGEALDGDSAYVSEYQGPLNSAINYPLWYPLIKAFMGGSFDELSAMISTEATAFSDVNALTNFLDNHD
QPRIASVAGDDEVRDKNAVTFLMFTSGIPMVYYGFEQRFSGAADPDNRETLWTSGYDTTTALYQHIAKLHEIRGVASNVTDKATYFSSNVAVLGTSTAYMAIERGPIVAVVSNVGAAGTSDGFDVTGSKFASGDSVADLLDCATTATVGDSGAFTSPSNNGEARIWIQAENKGSLCA
This Geomyces pannorum α-amylase aminoacid sequence is 58% with the highest similarity of α-amylase aminoacid sequence of having reported.Therefore this gene is a new alpha-amylase gene, by it called after GpA2.
Although more than provide and obtained the method for total length alpha-amylase gene DNA and cDNA from wild strain by PCR method, but, those skilled in the art's gene order disclosed according to the present invention, adopt other method, as synthetic method, can obtain total length alpha-amylase gene DNA and cDNA equally, this is apparent.
The structure of the aspergillus oryzae recombinant expression vector that embodiment 2. contains low temperature alpha-amylase gene
2.1, the genomic extraction of aspergillus oryzae
Aspergillus oryzae Aspergillus oryzae RIB40 (National Research Institute of Brewing Stock Culture and ATCC42149) is inoculated on CM substratum, and at 30 ℃, constant temperature culture 7 days is ripe to spore.Prepare appropriate spore suspension and be inoculated in CM medium liquid seed culture medium, cultivate and reach 4～5g/L for extracting genome to mycelium concentration in 2 days under 30 ℃ of 200rpm conditions, method is extracted test kit specification sheets with reference to Omega fungal gene group
2.2, the separation of aspergillus oryzae α-amylase promotor (PamyB) and signal peptide sequence thereof
According to the sequence of the amyB promotor of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primer PaXf and downstream primer Pamr, carries out the separated PaS of special pcr amplification (PamyB+signal peptide).Wherein
The sequence of PaXf is SEQ ID NO.20:CCG
cTCGAGgAATTCATGGTGTTTTGATCATTTT, wherein contains XhoI restriction enzyme site
The sequence of Pamr is SEQ ID NO.21:A
gGCGCGCCGCTAGCaGGCGTTGCAGCCAAAGC, wherein contains AscI and NheI restriction enzyme site
2.3, the separation of aspergillus oryzae α-amylase terminator (TamyB)
According to the sequence of the amyB terminator of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primer TaNf and downstream primer TaNr, carries out the separated PaS of special pcr amplification (PamyB+signal peptide).Wherein
The sequence of TaNf is SEQ ID NO.22:ATAAGAAT
gCGGCCGCgGGTGGAGAGTATATG, wherein contains NotI restriction enzyme site
The sequence of TaNr is SEQ ID NO.23:ATAAGAAT
gCGGCCGCaATTCTTGAGGACCATTAC, wherein contains NotI restriction enzyme site
2.4, the separation of low temperature alpha-amylase gene
The pMD19T simple that has connected GpA2 of take is template, uses primer 12A2mf and 12A2Hr to obtain GpA2 gene through pcr amplification.Wherein
The sequence of 12A2mf is SEQ ID NO.24:GCCT
gCTAGCGGCGCGCCtAAAACCGCCGCGGAATGG, wherein contains AscI and NheI restriction enzyme site
The sequence of 12A2Hr is SEQ ID NO.25:CCC
aAGCTTtTAAGCACATAAACTGCCCT, wherein contains HindIII restriction enzyme site
2.5, the separation of aspergillus oryzae orotidine phosphoribosyltransferase selection markers gene
According to the sequence of pyrF in the full genome of aspergillus oryzae RIB40 of the upper issue of GeneBank, the aspergillus oryzae genomic dna of said extracted of take is template, with upstream primers F sNf and downstream primer FxNr, carries out the separated pyrF expression cassette of special pcr amplification.Wherein
The sequence of FsNf is SEQ ID NO.26:GGAATTC
cATATGtGAAAGACTGCTGCAAAGCC, wherein contains NdeI restriction enzyme site
The sequence of FsNr is SEQ ID NO.27:GGAATTC
cATATGaAGCAGTCGTACATACATGG, wherein contains NdeI restriction enzyme site
All pcr amplification reaction conditions and reaction system, with reference to Vazyme high-fidelity enzyme reagent kit specification sheets, are cut glue purification with reference to Axygen test kit specification sheets above.PCR product is carried out to agarose gel electrophoresis, reclaim, connect pEASY-Blunt clonning vector, bacterium colony PCR checking positive colony send order-checking, and the biological order-checking of the farsighted enlightening that checks order company completes.
2.6, PaSA2 merges the structure of fragment
PaS and the GpA2 obtaining after PCR separation and purification of take is template, with upstream primer PaXf and downstream primer 12A2Hr, merges pcr amplification, obtains PaSA2 fragment, and introduces restriction enzyme site NheI and AscI.Its reaction system is:
Merge PCR reaction conditions with reference to Vazyme high-fidelity enzyme reagent kit specification sheets, cut glue purification with reference to Axygen test kit specification sheets.PCR product is carried out to agarose gel electrophoresis, reclaim, connect pEASY-Blunt clonning vector, bacterium colony PCR checking positive colony send order-checking, and the biological order-checking of the farsighted enlightening that checks order company completes.
2.7, the structure of aspergillus oryzae pBC12FA2 expression vector
Take pBC-Hygro as original plasmid, utilize the restriction enzyme site that its top carries that above-mentioned fragment is connected successively and builds pBC12FNHA2.Digestion with restriction enzyme and the order of connection are as follows: NotI is single endonuclease digestion TamyB and pBC-Hygro respectively, obtain pBC12 after connection; NdeI is single endonuclease digestion pyrF and pBC12 respectively, obtains pBC12F after connection; XhoI and HindIII be double digestion pBC12F and PaSA2 respectively, obtains pBC12FA2 after connection.DNA ligation completes connecting fluid is proceeded to intestinal bacteria competence DH5 α (Tian Gen company), and the LB that then coating contains paraxin (50mg/ml) is dull and stereotyped.Choose mono-clonal, the LB liquid nutrient medium that access contains paraxin (50mg/ml), bacterium colony PCR checking positive colony send order-checking.After enlarged culturing, plasmid extraction is with reference to the little plasmid kit of carrying of sky root, obtains expression vector pBC12FA2, obtain can expression alien gene GpA2 aspergillus oryzae expression vector.
2.8, aspergillus oryzae pBC12FNHA2 can nucleophilic purification Table reaches the structure of carrier
Take pBC12FA2 as template, utilize KOD rite-directed mutagenesis test kit after signal peptide, insert the His-Tag label that is usually used in nucleophilic purifying before NheI and AscI, this sequence label is: CATCATCACCATCACCAT.Primer for the insertion of fixing a point is FA2NHf and FA2NHr.Its sequence is respectively
The sequence of FA2NHf is SEQ ID NO.28:
cATCACCATgCTAGCGGCGCGCCTAAAAC
The sequence of FA2NHr is SEQ ID NO.29:
gTGATGATGaGGCGTTGCAGCCAAAGCAG
Its operation steps is with reference to KOD rite-directed mutagenesis test kit specification sheets.Its subsequent step is as described in 2.6.Obtain can purifying foreign protein GpA2 expression vector pBC12FNHA2.
Fig. 1 shows pBC12FNHA2 expression plasmid building process.
Embodiment 3. is containing the conversion of the aspergillus oryzae recombinant bacterium of low temperature alpha-amylase expression vector
The preparation of 3.1 aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplastiss
(1) by A.oryzae RIB40-PF1, (bacterial strain RIB40-PF1 used in the present invention is that contriver oneself screening obtains, this bacterial strain has been preserved in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC NO:9129, and preservation day is 2014.05.08.) spore inoculating on inclined-plane is in CM liquid nutrient medium, 30 ℃ of 200rpm incubated overnight (12～16h), form fine and close very little bacterium ball (d<2mm), with under aseptic washing, with aseptic Microcloth, filter substratum and obtain mycelium, with solution I washing 2～3 times.In washing process, 4 ℃ of centrifugal 1min of 6000rpm, remove supernatant.Finally obtain clean mycelium (approximately 200mg).
(2) enzyme liquid system (5ml): cellulase 0.1g, helicase 0.1g, lywallzyme 0.025g.Filtration sterilization.
(3) in (1), in mycelium, add the enzyme liquid in 1ml (2), 150rpm, 30 ℃ of enzymolysis 3h.
(4) after enzymolysis, 4 ℃ of centrifugal 5min of 3500rpm, remove supernatant, solution II washing 1～2 time.
(5) add appropriate solution II (about 1ml) suspension protoplastis.
3.2 aspergillus oryzae uracil auxotrophy RIB40-PF1 protoplast transformation pBC12FNHA2
The 200ul protoplastis making in (1) 2.3 mixes with 1ng (about 20ul) plasmid pBC12FNHA2, adds 50ul solution III, ice bath 30min.
(2) in (1), in system, add 2ml solution III, room temperature is placed 5min.
(3) in (2), in system, add 4ml solution II, 4 ℃ of centrifugal 5min of 5000rpm, remove supernatant, and 400ul solution II suspends.
(4) JiangMM lower floor substratum is down flat plate, solidifies; MM upper strata substratum is down to 40 ℃ of left and right, sneaks into protoplast transformation liquid in (3), rocks rapidly flat board, and evenly tiling is placed 3～5d for 30 ℃.
The evaluation of 3.3 transformants and the expression of goal gene
The genomic dna of the positive transformant of extraction embodiment 3.2 steps (4) gained, as template, carries out pcr amplification with Auele Specific Primer pBCf and pBCr.
The sequence of FA2NHf is SEQ ID NO.30:TACTAGCAAGGGATGCCATGCTTGG
The sequence of FA2NHr is SEQ ID NO.31:ACTCAAACTCACTGTCCAATGCCAG
Result shows that the amplified production of transformant obtains the special band of target gene size, and is that template is carried out same PCR reaction with the genomic dna of original strain A.oryzaeRIB40-PF1, does not occur amplified production.
Abduction delivering and the purifying thereof of embodiment 4. recombinant alpha-amylases
The sub-abduction delivering of 4.1 recombinant conversion and screening
At recombinant bacterium slant culture 7d described in 2.5, scraping spore inoculating liquid fermentation medium, 20 ℃, 200rpm cultivates 6d.Suction filtration is collected supernatant, surveys enzyme and lives, and selects high enzyme transformant alive and carries out follow-up fermentation research.
The purifying of 4.2 recombinant alpha-amylases
By 4.1 fermentation culture suction filtrations, obtain crude enzyme liquid, by 5 times of its ultrafiltration and concentration (10kDa film), utilize GE HisTrap1ml pillar purified fermentation broth, measure enzyme and live, protein concentration, and detect with SDS-PAGE.
Fig. 2 shows the protein electrophoresis figure of Geomyces pannorum α-amylase aspergillus oryzae RIB40-PF1 recombinant bacterium abduction delivering.1 is albumen marker, size as figure mark, and 2 is the restructuring GpA2 after purifying, 3 is the crude enzyme liquid after concentrated, the 4 thick fermented liquids for restructuring transformant, the 5 thick fermented liquids for contrast host
Embodiment 5. α-amylase enzyme activity determinations
α-amylase enzyme activity determination adopts Yoo improved method
5.1 Specification Curve of Increasing
(1) Zulkovsky starch is first dried to constant weight, accurately takes 1.0000g, with deionized water, be settled to 1000ml, be made into 1g/l reference liquid;
(2) get the standard starch solution of 0,0.2,0.4,0.6,0.8,1.0ml, add pure water to 9.8ml;
(3) add 0.2ml0.2ml I
2-KI (I
2, 0.08% (w/v); KI, 0.8% (w/v)) solution colour developing, shake up, blue solution is stablized after 10min, at spectrophotometer wavelength 660nm place, measure light absorption value.
5.2 enzyme activity determination
(1) 1.000g Zulkovsky starch is dissolved in the 100ml citrate buffer (100mM, pH5.0) that is heated to boiling, is settled to 100ml after cooling.
(2) add 0.5ml substrate solution, 40 ℃ of preheating 10min, add the suitably enzyme liquid of dilution of 0.5ml, reaction 10min;
(3) add 0.2ml I
2-KI, shakes up, and blue solution is stablized after 10min, measures light absorption value at spectrophotometer wavelength 660nm place.
5.3 enzymes unit definition alive
Enzyme activity unit is defined as: a α-amylase Mei Huo unit (U) is defined as under specified criteria, the required enzyme amount of hydrolysis 1mg starch in 5min.
Embodiment 6. low temperature alpha-amylase zymologic properties are measured
The impact of 6.1 temperature on GpA2 enzyme activity
Under pH5.0 (100mM citric acid-sodium citrate damping fluid) condition, at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, measure Geomyces pannorum α-amylase enzyme activity respectively, the high enzymatic activity of take is 100%, calculates the enzyme activity under other temperature condition.
With the damping fluid of pH5.0, enzyme liquid is suitably diluted, respectively 40 ℃, 50 ℃, 60 ℃ insulations, after 5min, every 5min, sample (to 30min), cooling rapidly on ice, at 40 ℃, measure residual enzyme activity, take that there is no the enzyme liquid enzyme activity that heat is hatched be 100%, calculate enzyme activity, represent the thermostability of enzyme.
Fig. 3 shows that Geomyces pannorum α-amylase GpA2 optimum temperature is 40 ℃, and still keeps high enzyme vigor at 0-30 ℃, belongs to low-temperature amylase.
Fig. 4 shows that Geomyces pannorum α-amylase GpA2 is stable under 40 ℃ of conditions, and under 60 ℃ of conditions, hatches the basic inactivation of 5min, has embodied the thermal lability of cold-adapted enzyme.
The impact of 6.2pH on GpA2 enzyme activity
At 40 ℃, respectively under the condition of pH2.0, pH3.0 (glycine-hydrochloric acid), 4.0,5.0,6.0 (citric acid-sodium citrate), 7.0 (potassium primary phosphate-dipotassium hydrogen phosphates), 8.0 (Tris-hydrochloric acid), 9.0 (glycine-sodium hydroxide), measure Geomyces pannorum α-amylase enzyme activity, the high enzymatic activity of take is 100%, calculates the enzyme activity under other temperature condition.
With above-mentioned different damping fluids, enzyme liquid is suitably diluted, under 30 ℃ of conditions, hatch 30min respectively, take out coolingly rapidly on ice, under pH5.0,40 ℃ of conditions, measure residual enzyme activity, the enzyme liquid enzyme activity of not hatching of take is 100%, calculates the pH tolerance that enzyme activity represents enzyme.
Fig. 5 shows that the suitableeest action pH of Geomyces pannorum α-amylase is 5.0, has high enzyme vigor in interval, pH3.0～6.0.
Fig. 6 shows that Geomyces pannorum α-amylase all has good pH stability in the interval of pH4.0～9.0.
Above-described, be only preferred embodiment of the present invention, not in order to limit scope of the present invention.Simple, the equivalence that every claims according to the present patent application and description are done changes and modifies, and all falls into the claim protection domain of patent of the present invention.The present invention not detailed description be routine techniques content.
Claims (8)
1. from a low temperature alpha-amylase for fungi, be following (a) or protein (b): the protein (a) being formed by the aminoacid sequence shown in SEQ ID NO.19; (b) by the amino acid residue sequence of SEQ ID NO.19 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have α-amylase function by (a) derivative protein.
2. the encoding gene of a low temperature alpha-amylase according to claim 1.
3. encoding gene according to claim 2, is characterized in that, described encoding gene is the deoxyribonucleotide of following (1) or (2):
(1) there is nucleotide sequence shown in SEQ ID NO.17;
(2) there is nucleotide sequence shown in SEQ ID NO.18.
4. a recombinant expression vector, is characterized in that, this recombinant expression vector comprises according to the encoding gene described in claim 2 or 3.
5. recombinant expression vector according to claim 4, is characterized in that, described recombinant expression vector be take pBC-Hygro as original plasmid.
6. a recombinant host cell, is characterized in that, this recombinant host cell comprises recombinant expression vector according to claim 4.
7. recombinant host cell according to claim 6, is characterized in that, described recombinant host cell is that recombinant expression vector according to claim 4 is transferred to the recombinant host cell obtaining in the uracil auxotrophy bacterial strain of aspergillus oryzae.
8. recombinant host cell according to claim 7, it is characterized in that, described recombinant host cell is that recombinant expression vector according to claim 4 is transferred to preserving number is the recombinant host cell obtaining in the uracil auxotrophy bacterial strain RIB40-PF1 of aspergillus oryzae of CGMCC NO:9129.
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| CN105420214A (en) * | 2016-01-26 | 2016-03-23 | 华东理工大学 | Alpha-amylase and encoding gene thereof and application |
| CN110582573A (en) * | 2017-03-30 | 2019-12-17 | 南京百斯杰生物工程有限公司 | Expression of phytase in aspergillus niger |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105420214A (en) * | 2016-01-26 | 2016-03-23 | 华东理工大学 | Alpha-amylase and encoding gene thereof and application |
| CN105420214B (en) * | 2016-01-26 | 2018-10-09 | 华东理工大学 | A kind of alpha-amylase and its encoding gene and application |
| CN110582573A (en) * | 2017-03-30 | 2019-12-17 | 南京百斯杰生物工程有限公司 | Expression of phytase in aspergillus niger |
| CN110582573B (en) * | 2017-03-30 | 2023-10-31 | 南京百斯杰生物工程有限公司 | Phytase expression in Aspergillus niger |
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Application publication date: 20141203 |