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Coding gene of alpha-L-rhamnosidase mutant and expression vector of coding gene

A technology of rhamnosidase and coding gene, which is applied in the fields of glycosylase, genetic engineering, plant gene improvement, etc., to achieve the effect of improving enzyme activity and heat resistance stability

Active Publication Date: 2020-08-25
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of strains producing high-temperature-resistant α-L-rhamnosidase for industrial production, and the research on the breeding of high-temperature-resistant α-L-rhamnosidase-producing bacteria is extremely important for realizing the industrial production of naringinase

Method used

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  • Coding gene of alpha-L-rhamnosidase mutant and expression vector of coding gene
  • Coding gene of alpha-L-rhamnosidase mutant and expression vector of coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: Obtaining the coding gene of sequence mutation α-L-rhamnosidase mutant

[0014] In order to improve the temperature stability of wild-type α-L-rhamnosidase, the applicant conducted point mutation screening on the wild-type α-L-rhamnosidase gene (GenBank: AF284762.1), and finally screened for temperature stability Increased sequence mutation gene. The mutated nucleotide sequence is shown in SEQ ID NO:1. The wild-type α-L-rhamnosidase gene and the sequence of SEQ ID NO: 1 with restriction sites (EcoR I and Not I) were synthesized by chemical synthesis method.

Embodiment 2

[0015] Embodiment 2: Expression of sequence mutation α-L-rhamnosidase mutant gene

[0016] The wild-type α-L-rhamnosidase gene, the sequence mutation α-L-rhamnosidase mutant gene and the pPIC9K vector were cut with EcoR I and Not I respectively, and the wild-type α-L-rhamnosidase Gene and sequence mutation The α-L-rhamnosidase mutant gene was connected to the EcoR I and Not I sites of the pPIC9K vector to obtain recombinant vectors pPIC9K-R (containing the wild-type α-L-rhamnosidase gene) and pPIC9K -RM (alpha-L-rhamnosidase mutant gene containing sequence mutation). Transfer the recombinant vector pPIC9K-R into Pichia pastoris GS115 to obtain recombinant Pichia RhaO containing the wild-type α-L-rhamnosidase gene, and transfer the recombinant vector pPIC9K-RM into Pichia GS115 to obtain sequence mutation α-L - Recombinant Pichia RhaR of the rhamnosidase mutant gene.

[0017] Recombinant Pichia pastoris RhaO and recombinant Pichia pastoris RhaR were inoculated into 50mL liqui...

Embodiment 3

[0019] Example 3: Temperature stability analysis of α-L-rhamnosidase mutants

[0020] The enzyme-producing supernatants of the recombinant Pichia pastoris strain containing the original α-L-rhamnosidase gene and the recombinant Pichia pastoris strain containing the mutant α-L-rhamnosidase mutant gene in Example 3 were respectively Store and incubate at 35°C, 45°C, 55°C, 65°C, 75°C and 85°C for 10 minutes, take samples to measure α-L-rhamnosidase activity, take the initial α-L-rhamnosidase activity as 100 %Calculation of relative enzyme activity, the result shows ( figure 1 and figure 2 ) The heat-resistant stability of the α-L-rhamnosidase mutant of the present invention is significantly improved, and after being incubated at 65° C. for 10 min, it can still maintain 28% of the enzyme activity, while the wild-type α-L-rhamnosidase Only 13% of the enzyme activity remained.

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Abstract

The invention discloses a coding gene of an alpha-L-rhamnosidase mutant. The nucleotide sequence of the alpha-L-rhamnosidase mutant is shown as SEQ ID NO:1. According to the invention, site mutation is carried out on the basis of an aspergillus aculeatus wild type alpha-L-rhamnosidase gene; the similarity between the mutated alpha-L-rhamnosidase mutant gene and the wild type alpha-L-rhamnosidase gene is 99.89%; and compared with the wild type alpha-L-rhamnosidase mutant gene, after being expressed in pichia pastoris, the mutated alpha-L-rhamnosidase mutant gene has the advantages that enzyme activity and heat resistance of alpha-L-rhamnosidase are obviously improved. According to the method, catalytic performance and stability of the alpha-L-rhamnosidase under a high-temperature conditioncan be improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a sequence-improved α-L-rhamnosidase mutant coding gene and its expression carrier. Background technique [0002] Citrus juice is a natural juice obtained by pressing citrus fruits such as oranges, tangerines, and pomelo. It mainly contains pulp, protopectin, and fiber. However, the squeezed citrus juice generally has a bitter taste, which reduces the quality of the juice. The reason for the bitter taste of citrus juices is due to the bitter ingredients contained in them, the most important of which is naringin. Naringin, also known as naringin, citrusin and isohesperidin, is a kind of dihydroflavonoid compound. The peel, pulp and seeds of citrus fruits contain naringin. Among them, naringin in the peel highest content. During the processing of citrus fruit juice, naringin from various parts will inevitably be released into the juice, which will eventually lead to a bitter tast...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2402C12N15/815C12Y302/0104
Inventor 顾斌涛熊大维黄国昌金丹凤黄筱萍李鹏刘兰
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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