CN106834249B - A kind of thermostabilization inorganic pyrophosphatase of transformation - Google Patents
A kind of thermostabilization inorganic pyrophosphatase of transformation Download PDFInfo
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Abstract
The invention discloses a kind of thermostabilization inorganic pyrophosphatases, it is characterized in that, the pyrophosphatase is from the inorganic pyrophosphatase transformation that sequence is SEQ ID NO:1, and remodeling method is at least one of following methods: 1) transforming the 6th amino acids T as amino acid S；2) the 11st amino acids E is changed to amino acid K；3) the 72nd amino acids L is changed to amino acid I；4) the 73rd amino acids V is changed to amino acid I；5) it transform the 124th amino acids D as amino acid A；6) it transform the 160th amino acids A as Q or K.After transformation, the inorganic pyrophosphatase of acquisition has higher amplification efficiency, and thermal stability is also more preferable.
Description
Technical field
The present invention relates to the transformation of enzyme, in particular to a kind of transformation of thermostabilization inorganic pyrophosphatase.
Background technique
In vivo, the biosynthesis that NTPs/dNTPs participates in nucleic acid can generate by-product pyrophosphate, exist in vivo
Pyrophosphate can be decomposed under the catalytic action of pyrophosphatase, reduce pyrophosphoric acid and accumulate the negative effect that may cause.PCR benefit
With the vitro reactions of Zimadzhunt L 340 simulation nucleic acid in vivo duplication, with participating in for dNTPs, the accumulation of pyrophosphoric acid can equally reduce PCR
The efficiency of amplification.
The accumulation of pyrophosphoric acid, will lead to reaction mixture pH reduce, influence fluorescent dye stability (Roche,
200510099911.5).In order to remove inhibition of these pyrophosphoric acids to PCR, people add inorganic pyrophosphate in PCR reaction
Enzyme, pyrophosphoric acid (PPi) can be hydrolyzed to orthophosphoric acid (Pi).
This reaction, the fluorescent dye that the pH of reaction mixture can be made to increase, and be become more stable.
In order to adapt to the high temperature of PCR, this pyrophosphatase must be resistant to 95 DEG C of high temperature.
Zhao Dantong et al. has recombinantly expressed the PPase gene (Zhao Dan of hyperthermophilic archaeal (Pyrococcus horikoshii)
Expression, purifying and zymologic property research [D] the Jilin University of red recombination hyperthermophilic inorganic pyrophosphatase, 2003.), this enzyme
Optimum temperature is 88 DEG C, and optimal pH 10.3 is not appropriate for the use of PCR.
Mu Hang et al. has recombinantly expressed the PPase gene of HB27 plants of thermus thermophilus (Thermus thermophiles)
(Mu Hang, Xia Yong, Wu Xueqiang wait the expression and purification of heat resistant inorganic pyrophosphatase to beg for property research [C] // whole nation zymetology science
By meeting and 85 anniversary of Zou Chenglu birthday meeting .2008.), Wang Lei et al. has recombinantly expressed HB8 plants of thermus thermophilus of PPase
Gene.For the optimum temperature of this enzyme at 65 DEG C, optimal pH 8.0 is suitble to the use of PCR.However half at 95 DEG C of this enzyme
Decline phase only 20min, is insufficient for the PCR reaction that the initial denaturation time is longer and recurring number is more.
PPase gene based on thermus thermophilus as a result, constructs a kind of thermostabilization inorganic pyrophosphatase of transformation, this
Enzyme can be applied to need the super quick PCR amplification of more recurring numbers and reaction time, increase the amplification efficiency of reaction.
Summary of the invention
The purpose of the present invention is to provide a kind of new thermostabilization inorganic pyrophosphatases with higher amplification efficiency.
The technical solution used in the present invention is:
A kind of thermostabilization inorganic pyrophosphatase changes from the inorganic pyrophosphatase transformation that sequence is SEQ ID NO:1
Making method is at least one of following methods:
1) it transform the 6th amino acids T as amino acid S；
2) the 11st amino acids E is changed to amino acid K；
3) the 72nd amino acids L is changed to amino acid I；
4) the 73rd amino acids V is changed to amino acid I；
5) it transform the 124th amino acids D as amino acid A；
6) it transform the 160th amino acids A as Q or K.
A kind of thermostabilization inorganic pyrophosphatase, sequence are as follows:
MANLKSLPVGKKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGD
PLDGIVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALE
AKKGKWVRVTGWRDRQAALEEVKACIARYGK(SEQ ID NO:2)。
A kind of thermostabilization inorganic pyrophosphatase, sequence are as follows:
MANLKTLPVGEKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGD
PLDGLILSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALE
AKKGKWVRVTGWRDRKAALEEVKACIARYGK(SEQ ID NO:3)。
The beneficial effects of the present invention are: improved inorganic pyrophosphatase has higher amplification before transformation
Efficiency, thermal stability are also more preferable.
Detailed description of the invention
Fig. 1 is that pyrophosphatase expands lab diagram；Wherein a is 1 amplification curve of inorganic pyrophosphatase, and b is that inorganic pyrophosphate does not have 2
Amplification curve, c are inorganic pyrophosphatase amplification curve before being transformed, and d is the amplification curve for not adding inorganic pyrophosphatase.
Specific embodiment
The transformation of embodiment thermostabilization inorganic pyrophosphatase
The thermostabilization inorganic pyrophosphatase (MCLAB, article No. TL-100) of commercialization, sequence are as follows: MANLKTLPVGEKAP
EVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGLVLSTYPLLPGVVVEVRV
VGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIADVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWRDRAAALEEV
KACIARYGK(SEQ ID NO:1)。
The site of above-mentioned enzyme is transformed, site is transformed and remodeling method is as follows:
It transform the 6th amino acids T of the sequence of protoenzyme as amino acid S；11st amino acids E is changed to amino acid K；72nd
Amino acids L is changed to amino acid I or the 73rd amino acids V is changed to amino acid I；124th amino acids D transform amino acid A as；
160th amino acids A transform Q or K as.
Obtain the following thermostabilization pyrophosphatase of two amino acid sequences:
Inorganic pyrophosphatase 1:
MANLKSLPVGKKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGD
PLDGIVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALE
AKKGKWVRVTGWRDRQAALEEVKACIARYGK(SEQ ID NO:2)。
Inorganic pyrophosphatase 2
MANLKTLPVGEKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGD
PLDGLILSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALE
AKKGKWVRVTGWRDRKAALEEVKACIARYGK(SEQ ID NO:3)。
According to the artificial synthesized corresponding base sequence of the amino acid sequence of above-mentioned pyrophosphatase, construction of expression vector is expanded,
Expression albumen is simultaneously purified.
The detection of experimental example transformation enzyme effect
The thermostabilization inorganic pyrophosphatase that will be transformed in this patent, and the thermostabilization inorganic pyrophosphatase of commercialization
(MCLAB, article No. TL-100), 0.05U, 0.5 μ L are diluted to 0.1U/ μ L with enzyme dialyzate.
Using the HBV DNA detection kit (state's tool infuses standard 20163400199) of Guangzhou Hai Lite, detection HBV virus is pressed
Following table is tested:
In addition to adding inorganic pyrophosphatase, operated by kit specification, using 7500 fluorescent PCR instrument of ABI into
Row amplification, experimental result are as shown in Figure 1, the results showed that, Ct value is 11.38 when pyrophosphatase 1 expands, when pyrophosphatase 2 expands
Ct value is 11.61, and Ct value is 15.55 when original thermostabilization inorganic phosphate enzymatic amplification, when amplification without adding inorganic phosphate enzyme
Ct value is 17.45.It can be seen that higher by improved inorganic pyrophosphatase amplification efficiency, thermal stability is more preferable.
SEQUENCE LISTING
<110>Guangzhou Supbio Bio-Technology Co., Ltd.
<120>the thermostabilization inorganic pyrophosphatase of a kind of transformation
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 175
<212> PRT
<213>inorganic pyrophosphatase
<400> 1
Met Ala Asn Leu Lys Thr Leu Pro Val Gly Glu Lys Ala Pro Glu Val
1 5 10 15
Val Asn Met Val Ile Glu Val Pro Arg Gly Ser Gly Asn Lys Tyr Glu
20 25 30
Tyr Asp Pro Gly Leu Gly Val Ile Lys Leu Asp Arg Val Leu Pro Gly
35 40 45
Ala Gln Phe Tyr Pro Gly Asp Tyr Gly Phe Ile Pro Ser Thr Leu Ala
50 55 60
Glu Asp Gly Asp Pro Leu Asp Gly Leu Val Leu Ser Thr Tyr Pro Leu
65 70 75 80
Leu Pro Gly Val Val Val Glu Val Arg Val Val Gly Leu Leu Leu Met
85 90 95
Glu Asp Glu Lys Gly Gly Asp Ala Lys Ile Ile Gly Val Val Ala Glu
100 105 110
Asp Gln Arg Leu Asp His Ile Gln Asp Ile Ala Asp Val Pro Glu Gly
115 120 125
Val Lys Gln Glu Ile Gln His Phe Phe Glu Thr Tyr Lys Ala Leu Glu
130 135 140
Ala Lys Lys Gly Lys Trp Val Arg Val Thr Gly Trp Arg Asp Arg Ala
145 150 155 160
Ala Ala Leu Glu Glu Val Lys Ala Cys Ile Ala Arg Tyr Gly Lys
165 170 175
<210> 2
<211> 175
<212> PRT
<213>inorganic pyrophosphatase
<400> 2
Met Ala Asn Leu Lys Ser Leu Pro Val Gly Lys Lys Ala Pro Glu Val
1 5 10 15
Val Asn Met Val Ile Glu Val Pro Arg Gly Ser Gly Asn Lys Tyr Glu
20 25 30
Tyr Asp Pro Gly Leu Gly Val Ile Lys Leu Asp Arg Val Leu Pro Gly
35 40 45
Ala Gln Phe Tyr Pro Gly Asp Tyr Gly Phe Ile Pro Ser Thr Leu Ala
50 55 60
Glu Asp Gly Asp Pro Leu Asp Gly Ile Val Leu Ser Thr Tyr Pro Leu
65 70 75 80
Leu Pro Gly Val Val Val Glu Val Arg Val Val Gly Leu Leu Leu Met
85 90 95
Glu Asp Glu Lys Gly Gly Asp Ala Lys Ile Ile Gly Val Val Ala Glu
100 105 110
Asp Gln Arg Leu Asp His Ile Gln Asp Ile Ala Ala Val Pro Glu Gly
115 120 125
Val Lys Gln Glu Ile Gln His Phe Phe Glu Thr Tyr Lys Ala Leu Glu
130 135 140
Ala Lys Lys Gly Lys Trp Val Arg Val Thr Gly Trp Arg Asp Arg Gln
145 150 155 160
Ala Ala Leu Glu Glu Val Lys Ala Cys Ile Ala Arg Tyr Gly Lys
165 170 175
<210> 3
<211> 175
<212> PRT
<213>inorganic pyrophosphatase
<400> 3
Met Ala Asn Leu Lys Thr Leu Pro Val Gly Glu Lys Ala Pro Glu Val
1 5 10 15
Val Asn Met Val Ile Glu Val Pro Arg Gly Ser Gly Asn Lys Tyr Glu
20 25 30
Tyr Asp Pro Gly Leu Gly Val Ile Lys Leu Asp Arg Val Leu Pro Gly
35 40 45
Ala Gln Phe Tyr Pro Gly Asp Tyr Gly Phe Ile Pro Ser Thr Leu Ala
50 55 60
Glu Asp Gly Asp Pro Leu Asp Gly Leu Ile Leu Ser Thr Tyr Pro Leu
65 70 75 80
Leu Pro Gly Val Val Val Glu Val Arg Val Val Gly Leu Leu Leu Met
85 90 95
Glu Asp Glu Lys Gly Gly Asp Ala Lys Ile Ile Gly Val Val Ala Glu
100 105 110
Asp Gln Arg Leu Asp His Ile Gln Asp Ile Ala Ala Val Pro Glu Gly
115 120 125
Val Lys Gln Glu Ile Gln His Phe Phe Glu Thr Tyr Lys Ala Leu Glu
130 135 140
Ala Lys Lys Gly Lys Trp Val Arg Val Thr Gly Trp Arg Asp Arg Lys
145 150 155 160
Ala Ala Leu Glu Glu Val Lys Ala Cys Ile Ala Arg Tyr Gly Lys
165 170 175
Claims (2)
1. a kind of thermostabilization inorganic pyrophosphatase, which is characterized in that the amino acid sequence of the inorganic pyrophosphatase are as follows: MANLK
SLPVGKKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGIVLSTYPLL
PGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWR
DRQAALEEVKACIARYGK(SEQ ID NO:2).
2. a kind of thermostabilization inorganic pyrophosphatase, which is characterized in that the amino acid sequence of the inorganic pyrophosphatase are as follows: MANLK
TLPVGEKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGLILSTYPLL
PGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWR
DRKAALEEVKACIARYGK(SEQ ID NO:3).
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| CN113481180A (en) * | 2021-07-05 | 2021-10-08 | 吉林大学 | Alkaline thermophilic inorganic pyrophosphatase and application thereof in enhancing polymerase chain reaction and UDP-galactose synthesis reaction |
| CN114507649B (en) * | 2022-02-16 | 2023-11-21 | 吉林大学 | Thermophilic enzyme and method for efficiently synthesizing UDP-glucose and UDP-glucuronic acid by one-pot method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011135041A1 (en) * | 2010-04-30 | 2011-11-03 | Roche Diagnostics Gmbh | System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus |
| CN105017399A (en) * | 2015-07-10 | 2015-11-04 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011135041A1 (en) * | 2010-04-30 | 2011-11-03 | Roche Diagnostics Gmbh | System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus |
| CN105017399A (en) * | 2015-07-10 | 2015-11-04 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 古细菌P.horikoshii无机焦磷酸酶的克隆、纯化以及酶学性质;卢冬梅，等;《中国生物化学与分子生物学会第八界会员***暨全国学术会议论文摘要集》;20010901;第318-319页 * |
| 球形红细菌无机焦磷酸酶的原核表达、纯化及初步分析;黄园波，等;《山西农业科学》;20130531;第41卷(第5期);第427-433页 * |
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