CN112553180A - Archaea high-temperature amylase and application thereof - Google Patents

Archaea high-temperature amylase and application thereof Download PDF

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CN112553180A
CN112553180A CN202011593188.7A CN202011593188A CN112553180A CN 112553180 A CN112553180 A CN 112553180A CN 202011593188 A CN202011593188 A CN 202011593188A CN 112553180 A CN112553180 A CN 112553180A
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amylase
amypap
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enzyme activity
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曾湘
邵宗泽
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Third Institute of Oceanography MNR
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Abstract

An archaea high-temperature amylase and application thereof, relating to amylase. By introducing deep-sea hyperthermophilic archaea pacific Pyrococcus (Palaececoccus pacific DY 20341)T) The amylase gene is transferred into Escherichia coli (Escherichia coli) after sequence optimization, an engineering strain for efficiently recombining and expressing the amylase is constructed, and the application range of the amylase is determined by analyzing the chemical characteristics of the recombinant protease. The archaea high-temperature amylase AmyPap gene is 1311bp in length, is nucleotide in type and has a sequence shown as SEQ ID No. 1. The archaea high-temperature amylase AmyPap has application range including pH, temperature, metal ions and the like. High temperature amylase AmyThe Pap can be applied to the industries of alcohol brewing, monosodium glutamate and starch sugar, but is not limited to the application in the food industry. The expressed protein has the characteristics of wide high-temperature action range and high thermal stability.

Description

Archaea high-temperature amylase and application thereof
Technical Field
The invention relates to amylase, in particular to archaea high-temperature amylase and application thereof.
Background
Amylases (amylases) are a generic term for a class of enzymes that degrade starch and glycogen, hydrolyzing only alpha-1, 4-glucosidic bonds in the starch molecular chain, cleaving the starch chain into short-chain dextrins, oligosaccharides and small amounts of maltose and glucose, which are widely found in plants, animals and microorganisms. The high-temperature amylase generally refers to amylase with the optimum enzyme activity temperature of more than 60 ℃, has the characteristics of strong thermal stability under the high-temperature condition, wide preservation condition, energy conservation, cost reduction, easy preservation and transportation and the like, and is widely applied to the industries of producing starch sugar (glucose, maltose, dextrin, fructose, oligosaccharide), alcohol, beer, monosodium glutamate, food brewing, fermentation industry, decontamination, textile and the like. Alpha-amylases which are used in large amounts in industry at present are mainly derived from Bacillus licheniformis (MEGAZYME, E-BLAAM), Bacillus amyloliquefaciens (MEGAZYME, E-BAASS), Bacillus subtilis subsp.str.168(PROZOMIX, PRO-E0403), Aspergillus niger and Aspergillus oryzae; the Bacillus licheniformis (MEGAZYME, E-BLAAM) is widely used due to high thermal stability, the optimum enzyme activity temperature is 75 ℃, the enzyme activity can be kept stable below 80 ℃, the optimum pH is 6.0-6.5, and the activity is in the range of pH 4.5-8.0.
The invention relates to a hyperphosphathermic archaea pacifica pathogenic bacterium DY20341TBelonging to the order of Thermococcus and isolated from the eastern Pacific volcanic hydrothermal region [1]. Pyrococcales are a common hyperthermophilic archaea in deep-sea hydrothermal regions, can utilize proteins and polysaccharides to perform heterotrophic metabolism, and have abundant carbohydrate degradation systems including degradation of starch, glycogen and polysaccharides. Thermophilic amylases derived from Thermococcus are reported to have thermophilic, acid-resistant properties, such as Pyrococcus furiouus, Pyrococcus woesei, Thermococcus ornurineus NA1, Thermococcus sp.HJ21.
Disclosure of Invention
The invention aims to provide an archaea high-temperature amylase AmyPap and application thereof. By introducing deep-sea hyperthermophilic archaea pacific Pyrococcus (Palaececoccus pacific DY 20341)T) The amylase gene is transferred into Escherichia coli (Escherichia coli) after sequence optimization, an engineering strain for efficiently recombining and expressing the amylase is constructed, and the application range of the amylase is determined by analyzing the chemical characteristics of the recombinant protease.
The molecular type of the archaea high-temperature amylase AmyPap gene is DNA, and the sequence characteristics are as follows: the length is 1311bp, the type is nucleic acid, the strand property is double-strand, and the topological structure is linear. The nucleotide sequence is obtained by the following method: obtaining a complete Open Reading Frame (ORF) by using genome analysis, after removing signal peptide and codon optimization, and carrying enzyme cutting sites NdeI and XhoI at the head and the tail of the sequence, synthesizing to obtain AmyPap which is marked as SEQ ID No.1, wherein the SEQ ID No.1 is as follows:
Figure BDA0002869703260000021
the molecular type of the high-temperature amylase AmyPap gene is protein, and the sequence characteristics are as follows: 453aa in length, amino acid type, sequence SEQ ID No.2, as follows:
Figure BDA0002869703260000022
Figure BDA0002869703260000031
an expression method of archaea high-temperature amylase AmyPap gene comprises the following steps:
1) constructing recombinant expression engineering bacteria of a high-temperature amylase AmyPap gene, wherein the vector is pET-28a-AmyPap, and the cultured cell is escherichia coli BL21(DE 3);
2) provides a method for expressing a large amount of high-temperature amylase AmyPap.
The archaea high-temperature amylase AmyPap has the application range including pH, temperature, metal ions and the like.
The pH range of the enzyme activity is pH4.5-9.5, and the optimum pH5.5-6.5; the temperature action range of enzyme activity is 55-99 ℃, and the optimum temperature is 60-80 ℃; it can endure long-time high-temperature treatment, and the enzyme activity can be independent of metal ions.
The high-temperature amylase AmyPap can be applied to the industries of alcohol brewing, monosodium glutamate and starch sugar, but is not limited to the application in the food industry.
The invention provides a hyperthermophilic from deep-sea hydrothermal areasPyrococcus pacificus Palaeococcus pacificus DY20341TThe expressed protein has the characteristics of wide high-temperature action range (50-99 ℃) and high thermal stability. The amylase AmyPap can keep more than 80% of enzyme activity at the temperature of 55-99 ℃, and has relatively high thermal stability. AmyPap Activity is Ca-independent2+，Mg2+And (4) plasma metal ions.
Drawings
FIG. 1 is SDS-PAGE electrophoresis chart of high temperature resistant amylase AmyPap gene recombination expression. The recombinant vector pET-28a-AmyPap is a strain of Escherichia coli BL21(DE 3). In FIG. 1, M is a protein Marker; line 1: total protein without induced expression; line 2: culturing the total protein of the supernatant after induction expression at 20 ℃; line 3: culturing and inducing the total protein of the precipitate after expression at 20 ℃; line 4: culturing the total protein of the supernatant after induction expression at 37 ℃; line 5: total protein precipitated after induction of expression in 37 degree culture.
FIG. 2 shows the enzymatic properties of the high temperature amylase AmyPap (influence of pH on the recombinant amylase AmyPap).
FIG. 3 shows the enzymatic properties of the high temperature amylase AmyPap (temperature effect on recombinant amylase AmyPap).
Detailed Description
The following examples will further illustrate the present invention with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the specific conditions not specified in the examples below were generally carried out according to conventional conditions such as those described in the molecular cloning laboratory manual (15, SammBruke, Lassel, Huang Petang (ed.), molecular cloning, A laboratory Manual, scientific Press, 2002, third edition) or according to the conditions recommended by the manufacturers of reagents or instruments.
In order to achieve the purpose, the invention adopts the following technical scheme, which comprises the following specific steps:
1. construction of high-temperature amylase AmyPap gene expression engineering bacteria
As the complete high-temperature amylase AmyPap is obtained by whole genome sequence analysis, a signal peptide sequence is removed, a certain company is entrusted to directly synthesize the AmyPap with enzyme cutting sites at two ends after codon optimization, NdeI and XhoI double enzyme cutting are respectively carried out on the synthesized AmyPap gene and pET-28a, enzyme cutting products of the two enzymes are purified and then connected overnight and transformed into escherichia coli Top10, 2-3 positive bacterial colonies are selected after colony PCR, bacteria are shaken, cultured and plasmids are extracted and subjected to sequencing detection. The positive clone expression plasmid was designated as pET-28 a-AmyPap. Escherichia coli BL21(DE3) was electrically transformed with the expression plasmid pET-28a-AmyPap, and a positive colony was selected as a recombinant strain and named BL21-pET-28 a-AmyPap.
2. Provides the expression condition of high-temperature amylase AmyPap
Inoculating the single colony of escherichia coli amylase engineering bacteria BL21-pET-28a-AmyPap into 10mL LB culture medium containing 50ug/mL kanamycin, and culturing at 37 ℃ until the bacterial liquid concentration OD600Up to 1.0. Inoculating 1mL of bacterial liquid into 100mL of LB culture medium containing 50ug/mL of kanamycin to culture until OD is reached600When the concentration reached 0.6, 0.5mM inducer IPTG was added, and the cells were cultured overnight at 20 ℃ or 37 ℃ for mass expression, and centrifuged to collect the cells. As shown in FIG. 1, after induction, a large amount of the protein of interest AmyPap was expressed.
After the centrifugal thalli are subjected to ultrasonic crushing, the supernatant is centrifugally collected, and purified protein is obtained by using a His-tagged nickel column. Adopting nickel NTA sepharose FF prepacked column, pre-eluting with 50mM imidazole-containing eluent, then obtaining high-purity target protein AmyPap with 500mM imidazole-containing eluent, dialyzing, concentrating with PEG20000, filtering with 0.45 μm filter membrane, packaging, and storing at-80 deg.C.
3. Providing application conditions of high-temperature amylase AmyPap
Amylase activity is characterized by measuring the amount of enzyme required for the hydrolysis of amylase to produce a reducing sugar. Using DNS reagent method, OD540And (4) comparing the detected color rendering value with a glucose standard curve, and calculating the content of reducing sugar in the reaction system. Buffer solutions (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0) with different pH values are used as reaction systems, the enzyme activity is measured at the temperature of 70 ℃, and the relative enzyme activity is calculated by taking the highest enzyme activity as 100 percent. As shown in figure 2, the amylase AmyPap of the invention has a pH value of 4.5-9.5The enzyme activity can be kept above 60% in the environment, and the optimal action is 5.5-6.5. The enzyme activity was measured at 37 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 99 deg.C, 110 deg.C, pH6.0, and the relative enzyme activity was calculated with the highest enzyme activity as 100%. As shown in figure 3, the amylase AmyPap can keep more than 80% of enzyme activity at the temperature of 55-99 ℃, and the optimal action temperature is 60-80 ℃. After the enzyme solution is respectively placed at the temperature of 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 99 ℃ for 4 hours, the residual enzyme activity is respectively 71.45 percent, 79.92 percent, 80.89 percent, 70.60 percent and 63.70 percent of the original enzyme activity, which shows that the enzyme solution has relatively high thermal stability. Adding different metal ions with final concentrations of 0.002M and 0.01M respectively into an optimal enzyme activity reaction system, and taking the enzyme activity without adding the metal ions as a reference (1.00); the results show (see Table 1), 0.01M Mg2+，Cu2+Has serious inhibiting effect on AmyPap, 0.01M Fe2+，K+Promoting the AmyPap activity; the addition of the chelating agent has no obvious influence on the enzyme activity, which indicates that the AmyPap activity does not depend on Ca2+，Mg2+And (4) plasma metal ions.
TABLE 1
Figure BDA0002869703260000051
Table 1 shows the effect of different metal ions and chelating agents on the enzymatic activity of the high-temperature amylase AmyPap.
Sequence listing
<110> third Marine institute of Natural resources department
<120> archaea high-temperature amylase and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1311
<212> DNA
<213> Pyrococcus pacificus (Palaecoccus pacificus DY20341T)
<400> 1
catatgctgt ctccagagga aggtggtgta atgatgcagg cattctactg ggacgtccct 60
gaaggtggta tctggtacga tactatccgt gcaaagatcc cggattgggc tgccgccggt 120
atcacttcta tctggctgcc tccaccatct aagggtatgt ctggtggtta ctccatgggt 180
tacgacccgt acgattactt cgacctgggt gagtactacc agatgggtac cgtcgagact 240
cgtttcggtt ctaagcagga gctgatcgat ctgatcaaca cggcacactc ttacggtctg 300
gaagtctacg cagacatcgt catcaaccac cgtgcaggtg gtgatctgga gtggaacccg 360
tttgtaaacg actacacctg gacggatttc tccaaagtgg catccggtaa atacaccgcg 420
aactacctgg acttccaccc gaacgaactg cacgcatctg atgctggtgc tttcggtggt 480
tacccggata tctgccatga taaaagctgg gaccagtact ggctgtgggc atcttctgaa 540
tcttacgcgg cttacctgaa aagcgtgggt ttcgacggtt ggcgtttcga ttacgtgaaa 600
ggctacgacc cgtgggtagt taaagactgg ctgtcctggt ggggtggtta tgctgttggt 660
gaatactggg ataccaacgt gaacctggtg ctggattgga tccgtggtag cggcgctaat 720
gctttcgatt tcgctctgta ctacaaaatg gacgaagctt tcgacaacac caacatcccg 780
gctctggtgt ccgcgattca aaacggccag gttctggttg cggttgatcc gtttgatgcg 840
gtaactttcg tggccaatca tgatacggat atcatctgga acaaataccc ggcctacgcg 900
tttatcctga cctacgaagg ccagccgact atcttctatc gcgactatga agaatggctg 960
aacaaagacc gcctgaccaa cctgatctgg attcacaaca acctggcggg cggcactacc 1020
gaaattgttt attatgacag cgacgaactg atttttgtac gcaacggcta tggctccaaa 1080
ccgggcctga ttacctatat taacctgggc tccggctggg cgggccgttg ggtttatgtt 1140
ccgaaatttg ccggcagcac cattcacgaa tataccggca acctgggcgg ctgggttgac 1200
aaatgggttg acagcaatgg ctgggtatat ctggaagcgc cgccgcatga cccggcgaat 1260
ggctattatg gctattccgt ttggagctat gcgggcattg gctaactcga g 1311
<210> 2
<211> 453
<212> PRT
<213> Pyrococcus pacificus (Palaecoccus pacificus DY20341T)
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Leu Ser Pro Glu Glu Gly Gly Val Met Met Gln
20 25 30
Ala Phe Tyr Trp Asp Val Pro Glu Gly Gly Ile Trp Tyr Asp Thr Ile
35 40 45
Arg Ala Lys Ile Pro Asp Trp Ala Ala Ala Gly Ile Thr Ser Ile Trp
50 55 60
Leu Pro Pro Pro Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly Tyr
65 70 75 80
Asp Pro Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr Gln Met Gly Thr
85 90 95
Val Glu Thr Arg Phe Gly Ser Lys Gln Glu Leu Ile Asp Leu Ile Asn
100 105 110
Thr Ala His Ser Tyr Gly Leu Glu Val Tyr Ala Asp Ile Val Ile Asn
115 120 125
His Arg Ala Gly Gly Asp Leu Glu Trp Asn Pro Phe Val Asn Asp Tyr
130 135 140
Thr Trp Thr Asp Phe Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn
145 150 155 160
Tyr Leu Asp Phe His Pro Asn Glu Leu His Ala Ser Asp Ala Gly Ala
165 170 175
Phe Gly Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr
180 185 190
Trp Leu Trp Ala Ser Ser Glu Ser Tyr Ala Ala Tyr Leu Lys Ser Val
195 200 205
Gly Phe Asp Gly Trp Arg Phe Asp Tyr Val Lys Gly Tyr Asp Pro Trp
210 215 220
Val Val Lys Asp Trp Leu Ser Trp Trp Gly Gly Tyr Ala Val Gly Glu
225 230 235 240
Tyr Trp Asp Thr Asn Val Asn Leu Val Leu Asp Trp Ile Arg Gly Ser
245 250 255
Gly Ala Asn Ala Phe Asp Phe Ala Leu Tyr Tyr Lys Met Asp Glu Ala
260 265 270
Phe Asp Asn Thr Asn Ile Pro Ala Leu Val Ser Ala Ile Gln Asn Gly
275 280 285
Gln Val Leu Val Ala Val Asp Pro Phe Asp Ala Val Thr Phe Val Ala
290 295 300
Asn His Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe
305 310 315 320
Ile Leu Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu
325 330 335
Glu Trp Leu Asn Lys Asp Arg Leu Thr Asn Leu Ile Trp Ile His Asn
340 345 350
Asn Leu Ala Gly Gly Thr Thr Glu Ile Val Tyr Tyr Asp Ser Asp Glu
355 360 365
Leu Ile Phe Val Arg Asn Gly Tyr Gly Ser Lys Pro Gly Leu Ile Thr
370 375 380
Tyr Ile Asn Leu Gly Ser Gly Trp Ala Gly Arg Trp Val Tyr Val Pro
385 390 395 400
Lys Phe Ala Gly Ser Thr Ile His Glu Tyr Thr Gly Asn Leu Gly Gly
405 410 415
Trp Val Asp Lys Trp Val Asp Ser Asn Gly Trp Val Tyr Leu Glu Ala
420 425 430
Pro Pro His Asp Pro Ala Asn Gly Tyr Tyr Gly Tyr Ser Val Trp Ser
435 440 445
Tyr Ala Gly Ile Gly
450

Claims (6)

1. An archaea high-temperature amylase is characterized in that the archaea high-temperature amylase AmyPap is derived from deep-sea hyperthermophilic archaea Palaecoccus pacificus DY20341TThe amylase gene is used for prokaryotic expression recombination nucleotide sequence after sequence optimization, the length is 1311bp, the type is nucleotide, and the sequence is shown as SEQ ID No. 1.
2. The archaea thermophilic amylase AmyPap gene as claimed in claim 1, which is characterized in that the molecular type is protein, and the sequence characteristics are as follows: the length is 453aa, the type is amino acid, and the sequence is shown as SEQ ID No. 2.
3. The method for expressing the archaea thermophilic amylase AmyPap gene as claimed in claim 1, which is characterized by comprising the following steps:
1) construction of recombinant expression vector pET-28 of high-temperature amylase AmyPapaAmyPap and recombinant expression engineering bacteria BL21-pET-28a-AmyPap；
2) Determining the high-efficiency expression condition of the recombinant high-temperature amylase AmyPap.
4. The archaea high-temperature amylase AmyPap as claimed in claim 1 is applied to the industries of alcohol brewing, monosodium glutamate and starch sugar, but not limited to the application in the food industry.
5. The use according to claim 4, characterized in that the conditions of the use are: the pH value of the enzyme activity ranges from 4.5 to 9.5, the temperature of the enzyme activity ranges from 55 to 99 ℃, and the enzyme activity does not depend on metal ions.
6. The use according to claim 5, characterized in that the conditions of the use are: the pH value of the enzyme activity is 5.5-6.5; the temperature range of enzyme activity is 60-80 ℃.
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JPH07143880A (en) * 1993-10-01 1995-06-06 Takara Shuzo Co Ltd Alpha-amylase gene having ultra-high heat-resistance
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CN101629180A (en) * 2009-07-02 2010-01-20 吉林大学 Thermophilic multifunctional amylase genes, thermophilic multifunctional amylases and application thereof
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CN104560792A (en) * 2014-12-15 2015-04-29 中国科学院微生物研究所 Amylase-producing strain and application thereof
US20190338264A1 (en) * 2018-05-04 2019-11-07 Fornia Biosolutions, Inc. Variant amylase enzyme compositions and methods
CN109182304A (en) * 2018-09-18 2019-01-11 云南中烟工业有限责任公司 A kind of alpha-amylase gene and its application
CN112029750A (en) * 2020-06-10 2020-12-04 华东理工大学 OUR-based fed-batch fermentation method for producing medium-temperature alpha-amylase by using bacillus subtilis

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