Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Recombinant pectinase, gene thereof, recombinant vector, preparation method and application

A pectinase and gene technology, applied in the field of biomolecule cloning, can solve problems such as being unsuitable for mass and low-cost production of pectinase, unsuitable for mass production of recombinant pectinase, inactivation of target products, etc. Safety, reducing the metabolic load of host cells, and achieving mass production effects

Active Publication Date: 2020-02-28
HUAIHUA UNIV
View PDF32 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Finally, obtaining high-purity proteins often requires multi-step purification operations. The more purification steps, the lower the yield of the protein, and it is more likely to cause the inactivation of the target product. Therefore, this expression system is not suitable for mass production of recombinant pectinase
Exogenous gene expression systems such as insect cell expression systems, plant expression systems, and mammalian expression systems have too high requirements for technology, equipment, and technical level, so the products produced are often expensive, and are also not suitable for large quantities of pectinases. low cost production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant pectinase, gene thereof, recombinant vector, preparation method and application
  • Recombinant pectinase, gene thereof, recombinant vector, preparation method and application
  • Recombinant pectinase, gene thereof, recombinant vector, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This embodiment provides an optimized artificially synthesized recombinant pectinase gene with a 6×His tag at the C-terminus. The specific sequence is shown in SEQ ID No.1 in the sequence listing, and the protein sequence corresponding to the gene As shown in SEQID No.2 in the sequence listing. The optimized DNA sequences were compared by NCBI, and there was no obvious similarity.

[0046] The present invention synthesizes the DNA sequence shown in SEQ ID No.1 according to the sequence characteristics of the pectinase gene itself and the yeast codon preference, the natural DNA of the pectinase before optimization, and the synthesized pectinase after optimization according to the codon preference of Escherichia coli The artificial DNA sequences were connected to the Pichia pastoris secretory expression vector pGAPZαA to obtain the recombinant vectors, and then the recombinant vectors were transformed into the Pichia pastoris host strain X-33 by using the lithium chloride...

Embodiment 2

[0048] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0049] S1: Construction of expression vector and transformation: The sequence characteristics of the gene itself and the DNA sequence synthesized by yeast codon preference in Example 1, that is, the DNA in SEQ ID No.1, were connected to the constitutive secretory expression vector pGAPZαA of Pichia pastoris to obtain Recombinant vector pGAPZαA-recombinant pectinase, the vector is constructed as figure 1 as shown, figure 1 It is a schematic diagram of the construction of the eukaryotic expression vector pGAPZαA-recombinant pectinase in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0050] (1) Digest the plasmid containing the synthetic recombinant pectinase gene (SEQ ID No.1) with Xho I and Xba I to obtain the target fragment. The reaction system is as follows (the endonuclease and buffer used were purchas...

Embodiment 3

[0081] In this example, the situation of improving the juice yield of apple, grape and orange juice by the purified recombinant pectinase is tested. The enzyme can increase the yield of fruit juice, the specific steps and results are as follows:

[0082] (1) 0, 1, 2, 4, 8, 16, 32 mg of recombinant pectinase are added to 100 grams of chopped apples respectively, and the apples with different concentrations of recombinant pectinase are fully Beat into pulp and let it stand at room temperature for 60 minutes; pour the apple pulp into 6 layers of gauze respectively, squeeze the juice out of the gauze until no juice flows out, and weigh them separately. The fruit juice weight that weighs is as shown in table 5, as can be seen, the fruit juice weight that the experimental group that has added pectinase obtains is all higher than the fruit juice amount that does not add recombinant pectinase produced, when adding 32 mg of recombinant pectinase When pectinase is added to 100 grams of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to recombinant pectinase, a gene thereof, a recombinant vector, a preparation method and application. The gene at least comprises DNA fragment of one of a nucleotide sequence ofSEQ ID NO.1 in a sequence table, a nucleotide sequence having higher than 90% homology with the nucleotide sequence shown as the SEQ ID NO.1 and coding same biological functional protein and a nucleotide sequence hybridized with the nucleotide sequence shown as the SEQ ID NO.1 and coding same biological functional protein. The recombinant vector is further built according to the gene sequence, yeast is transformed, in this way, constitutive secretory expression of the recombinant pectinase in a high-density fermentation condition can be realized; active protein higher than 95% in purity and with high protein concentration can be obtained through simple nickel affinity purification; the active protein has high pectin hydrolyzing activity at low temperature, and a novel and efficient enzymeproduct can be provided for increasing juice yield of fruit juice.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a recombinant pectinase and its gene, recombinant vector, preparation method and application. Background technique [0002] my country is a vast country with a land area of ​​about 9.6 million square kilometers. It spans the three major regions of the cold zone, temperate zone and subtropical zone in the north and south. It is suitable for the growth of many kinds of fruit trees. Therefore, my country is a big country in fruit production in the world, such as: hawthorn, apple, Pears, peaches, apricots, grapes, jujubes, seabuckthorns, black currants, kiwis, tangerines, oranges, tangerines, pineapples, bananas, guavas, prickly pears, passion fruit, etc. In the past ten years, my country's total fruit output has remained above 45 million tons. Rich fruit resources provide a good foundation for the development of my country's fruit juice industry. At present, the competit...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N9/24C12N9/88C12N15/55C12N15/56C12N15/60C12N15/81
CPCC12N9/18C12N9/2402C12N9/88C12N15/815C12Y301/01011C12Y302/01015C12Y402/02002C12N2800/22
Inventor 李洪波李露露何伶靖邓伟思董海丽
Owner HUAIHUA UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products