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Purine nucleoside phosphorylase and preparation method thereof

A purine nucleoside phosphorylase and polynucleotide technology, which is applied in the field of genetic engineering, can solve the problems of low PNP yield, inactivation, EcPNP changes sharply at 50-55 ℃, and the effect cannot be achieved.

Active Publication Date: 2013-12-25
AILEX TECH GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with PNP of other bacterial genera, PiPNP also has its disadvantages-lower temperature stability. When PiPNP is incubated for 30 minutes, the enzyme activity changes sharply at 37-42°C, and it is completely inactivated at 50°C. ℃ changes sharply, and the inactivation is completely lost at 65 ℃
[0005] In addition, according to various researches that have been reported, the yield of PNP produced by recombinant bacteria fermentation is still low, mostly 100-200U / ml. For industrial production, it still cannot achieve good results, and the yield needs to be improved.

Method used

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  • Purine nucleoside phosphorylase and preparation method thereof
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  • Purine nucleoside phosphorylase and preparation method thereof

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preparation example Construction

[0081] (3) The preparation method of the present invention greatly improves the output of purine nucleoside phosphorylase, and the expression activity of the product can reach or exceed 400U / mL, which is beyond the reach of the current genetic engineering preparation method. Thereby it is conducive to large-scale industrial production.

[0082] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments can be combined arbitrarily. All the features disclosed in the specification of this case can be used in combination with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

Embodiment 1

[0094] Example 1 Optimization of Pseudoalteromonas (Pseudoalteromonas) PNP gene sequence and construction of cloning vector

[0095] ① Optimization of gene sequence

[0096] The optimization of the gene sequence is to partially modify the PNP gene sequence (GEN BANK EF222283, SEQ ID NO.3) of Pseudoalteromonas sp.i590, which is divided into two parts: one is to convert the Arg2 and Leu24 encoding amino acids in the entire sequence , Leu77, Ile87, Leu134, Leu145 codons AGG, CTA, CTA, ATA, CTA, CTA were changed to CGA, CTG, CTG, ATC, CTG, CTG; the second is to transform the 97-position Asp into Tyr, and the codon is changed from GAC Change to TAT. The optimized gene sequence is used for whole gene synthesis.

[0097] Whole Gene Synthesis:

[0098] Entrust Shanghai Sangong Synthetic Co., Ltd. Firstly, the wild-type PNP gene sequence of Pseudoalteromonas sp.i590 was obtained from the NCBI database, and the codon of the 97th amino acid residue was changed from Asp97 to Tyr. In ...

Embodiment 2

[0120] Example 2 Construction of PNP(O)-pET-28a(+)-BL21 Expression Engineering Bacteria

[0121] ①Plasmid extraction

[0122] The positive clones with correct sequencing were inoculated into LBA medium, cultured with shaking at 37°C and 220 rpm for 12 hours, and the recombinant plasmid was extracted with BIOMIGA's Plasmid Miniprep Kit. For specific operation methods, please refer to the instruction manual.

[0123] ② enzyme digestion

[0124] Both PNP(O)-pMD19T-BL21 and the vector pET-28a(+) were digested with Nhe I and Xho I, overnight at 37°C; all digested products were used for agarose gel horizontal plate electrophoresis at a gel concentration of 1.0 %(W / V), based on TaKaRa's DL2000 TM DNA Marker was used as DNA molecular weight standard; after electrophoresis, the target fragment was recovered with UNIQ-10 Column DNA Gel Recovery Kit.

[0125] ③ connection

[0126] The connection system is shown in Table 4, and connected overnight at 16°C. The plasmid map of the resu...

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Abstract

The invention relates to purine nucleoside phosphorylase and a preparation method of the purine nucleoside phosphorylase, in particular to a mutational pseudoalteromonas-genus purine nucleoside phosphorylase. Compared with a wild-type amino acid sequence, the amino acid sequence of the mutational purine nucleoside phosphorylase is characterized in that Asp at the 98th bit is mutated into Tyr. The invention further discloses the preparation method of the purine nucleoside phosphorylase. The purine nucleoside phosphorylase and the preparation method of the purine nucleoside phosphorylase have the advantages of being high in yield and good in heat stability. The invention further discloses polynucleotide for encoding the amino acid sequence of the purine nucleoside phosphorylase, a carrier containing the polynucleotide and a host cell containing the polynucleotide.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a purine nucleoside phosphorylase and a preparation method thereof. Background technique [0002] Purine nucleoside phosphorylase (PNP for short), is one of the key enzymes in the purine salvage synthesis pathway, widely exists in mammals, parasites and microorganisms. According to the protein structure of purine nucleoside phosphorylase, it can be divided into two categories: low molecular weight homotrimers and high molecular weight homohexamers. Among them, the purine nucleoside phosphorylases of mammals and some microorganisms (such as Salmonella typhimurium, thermophilic archaea Sulfolobus solfataricus, etc.) belong to the homotrimer class, and its molecular weight is about 80-100kDa. The molecular weight of each subunit is 30-32kDa, and usually this kind of purine nucleoside phosphorylase can only use guanosine and inosine as substrates. The homohexamer purine nucleosid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12N15/10C12P17/18C12R1/01
Inventor 李子樵沈露
Owner AILEX TECH GRP CO LTD
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