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A method for improving the thermal stability of α-l-rhamnosidase r-rha1

A technology of rhamnosidase and thermal stability, applied in the fields of genetic engineering and enzyme engineering, to achieve the effect of excellent enzymatic properties

Active Publication Date: 2019-08-13
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few reports on α-L-rhamnosidase derived from animal tissues and plant tissues, mainly through bacteria such as Bacillus, Bacteroides, Lactobacillus, Streptomyces, and Fermentation of Aspergillus aculeatus, Aspergillus terreus, Penicillium, Trichoderma, yeast, Absidia and other fungi to obtain α-L-rhamnosidase

Method used

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  • A method for improving the thermal stability of α-l-rhamnosidase r-rha1
  • A method for improving the thermal stability of α-l-rhamnosidase r-rha1
  • A method for improving the thermal stability of α-l-rhamnosidase r-rha1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Acquisition of α-L-rhamnosidase gene

[0029] Escherichia coli DH5α containing WT (pPIC9K-rha) plasmid was inoculated in 30 mL LB liquid medium containing 1 mg / mL ampicillin resistance and cultured at 37 ° C for 16 h.

[0030] The WT (pPIC9K-rha) plasmid was extracted using the plasmid mini-extraction kit (Takara Company) according to the instructions. EcoRI and BlnI double enzyme digestion was used for verification. After verifying that the result is correct, it is used as a template for error-prone PCR.

Embodiment 2

[0031] Example 2: Construction and screening of α-L-rhamnosidase mutant library

[0032] The WT gene was subjected to error-prone PCR to establish a mutant library, and the enzyme was induced and expressed in a 96-microwell plate. After screening, a mutant with significantly improved thermal stability was obtained.

[0033] Using the WT (KC750908.1) gene, design a pair of specific primers:

[0034] Upstream primer Q9KF (SEQ ID NO.3): 5'-CCG GAATTC GTACCCTACGAGGAGTACATTTCTAG-3′,

[0035] Downstream primer Q9KR (SEQ ID NO.4): 5'-CGC CCTAGG TTACACATTCAACCGCCATTTC-3';

[0036] The PCR reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 2.5 min, and after 35 cycles, extension at 72°C for 7 min. PCR products were purified using Takara PCR Product Column Recovery Kit.

[0037] Recombinant expression plasmids were constructed by restriction cloning, that is, the error-prone PCR products...

Embodiment 3

[0039] Example 3: Expression and purification of α-L-rhamnosidase WT and V529A using recombinant expression strains

[0040] Inoculate the strains with 1% inoculum in 50mLYPD liquid medium for strain activation, shake culture at 30°C for 16h; min, after culturing for 16 hours, measure and determine its OD 600Reach the range of 3.0-5.0; centrifuge for 10 minutes to collect all the bacteria, discard the supernatant, transfer all the bacteria to 100mL BMMY medium, 30°C, culture for 7 days, add 0.5% to the medium every 24h during the culture period Sterile methanol solution; after cultivation, the supernatant collected by centrifugation is the enzyme solution.

[0041] The α-L-rhamnosidase WT and V529A crude enzyme solutions were collected, concentrated by ultrafiltration through a 30kDa membrane, and set aside. At a flow rate of 0.5mL / min, use 20mmol / L of C 6 h 8 o 7 -Na 2 HPO 4 buffer and 0.15mol / L sodium chloride solution on Sephacryl TM Equilibrate S-200HR, load the co...

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Abstract

The invention discloses a method for improving thermal stability of alpha-L-rhamnosidase gamma-Rha1. The method comprises the following steps: taking a wild-type alpha-L-rhamnosidase gamma-Rha1 (WT) gene as a template, performing random mutation by an error-prone PCR method to establish a mutant library of the alpha-L-rhamnosidase, and establishing a 96-pore plate expression-inducing and high-throughput screening method to eventually obtain a mutant with improved thermal stability. A V529A mutant and the WT have the same tolerance to a metal ion and an effector which are used in a test. The mutant V529A has excellent enzymological properties; meanwhile, by the method provided by the invention, a mutant V529A-containing recombinant vector is successfully established, heterologous expression is achieved, and a good foundation is provided for industrial production and application of the alpha-L-rhamnosidase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and enzyme engineering, in particular to a method for improving the thermal stability of alpha-L-rhamnosidase r-Rhal. Background technique [0002] α-L-rhamnosidase (α-L-rhamnosidase (EC 3.2.1.40)) belongs to the converting glycoside hydrolase class, which can specifically and efficiently hydrolyze many glycosides such as naringin (naringin), rutin ( rutin), hesperidin (hesperidin) and other terminal α-L-rhamnosyl groups. The classification of glycoside hydrolases in the CAZy (http: / / www.cazy.org / ) database is based on the similarity of their amino acid sequences. α-L-rhamnosidases are included in four glycoside hydrolase families (glycoside hydrolase family, GH), which are GH13, GH28, GH78 and GH106, respectively. [0003] The sources of α-L-rhamnosidase are very extensive, and there have been reports of α-L-rhamnosidase derived from animal tissues, plant tissues and microorganisms....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/81C12R1/84
CPCC12N9/2402C12Y302/0104
Inventor 李利君吴喆瑜倪辉杨远帆于越朱艳冰姜泽东肖安风
Owner JIMEI UNIV
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