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[Alpha]-L-rhamnosidase as well as preparation method and application thereof

A rhamnosidase and nucleotide sequence technology, applied in the field of alpha-L-rhamnosidase preparation, can solve the problems of low substrate conversion rate, low catalytic efficiency, long reaction time and the like, and achieve catalytic efficiency High, specific, and simple results

Pending Publication Date: 2021-11-02
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention is provided to make innovations and improvements based on the shortcomings of the existing rhamnosidase, such as low catalytic efficiency, low substrate conversion rate, and long reaction time.

Method used

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  • [Alpha]-L-rhamnosidase as well as preparation method and application thereof
  • [Alpha]-L-rhamnosidase as well as preparation method and application thereof
  • [Alpha]-L-rhamnosidase as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Prepare alpha-L-rhamnosidase CHD-R3 and study its enzymatic properties:

[0051] 1. Genome sequencing and gene function annotation of Basilicum bacteria

[0052] Use Solarbio's fungal genome extraction kit for Basilisk bacteria ( Talaromyces Stollii , Preservation Unit: CGMCC, General Microbiology Center of China Microbiological Culture Collection Management Committee, Preservation Number: CGMCC No.3.16013, Preservation Date: 2020.8.28) Genomes were extracted, and then the extracted genomes were analyzed using the Illumina SolexaGenomeAnalyzer next-generation sequencer , to obtain the raw data of the basket-shaped bacteria genome. Use FASTQC to evaluate the quality of the raw data generated by the sequencing, and use Trimmomatic to cut the quality of the Illumina sequencing data to obtain relatively accurate and effective data. The results show that the quality of the raw data measured is good, and the next step of splicing annotation can be performed .

[0053] SPA...

Embodiment 2

[0080] Expression and purification of alpha-L-rhamnosidase CHD-R3 double mutant (G159F / S316N)

[0081] Amino acid residues Gly159 and Ser316 in the sequence SEQ ID NO. 1 were mutated to Phe159 and Asn316 respectively by site-directed mutagenesis, and the mutated amino acid sequence is shown in SEQ ID NO. 5. In the synthetic sequence list, SEQ ID NO.3 and SEQ ID NO.4, wherein the 5' end of the primer sequence SEQ ID NO.3 is provided with Eco RI restriction site and its protected base sequence, the 3' end of the primer sequence SEQ ID NO. 4 is provided with not I Restriction site and its protected base sequence. The mutated alpha-L-rhamnosidase CHD-R3 nucleotide sequence was used as a template for PCR amplification.

[0082] use Eco RI and not The alpha-L-rhamnosidase CHD-R3 nucleotide mutant sequence obtained after I two kinds of restriction endonucleases will amplify and p The PIC9k vector was subjected to double enzyme digestion reaction at the same time, and passed ...

Embodiment 3

[0086] Expression and purification of alpha-L-rhamnosidase CHD-R3 triple mutant (A58S / Y91I / G159N)

[0087] Amino acid residues Ala58, Tyr91, and Gly159 in the sequence SEQ ID NO. 1 were mutated to Ser58, Ile91, and Asn159 by site-directed mutagenesis, and the mutated amino acid sequence is shown in SEQ ID NO. 6. In the synthetic sequence list, SEQ ID NO. 3 and SEQ ID NO. 4, wherein the 5' end of the primer sequence SEQ ID NO. 3 is provided with Eco RI restriction site and its protected base sequence, the 3' end of the primer sequence SEQ ID NO. 4 is provided with not I Restriction site and its protected base sequence. The mutated alpha-L-rhamnosidase CHD-R3 nucleotide sequence was used as a template for PCR amplification.

[0088] use Eco RI and not The alpha-L-rhamnosidase CHD-R3 nucleotide mutant sequence obtained after I two kinds of restriction endonucleases will amplify and p The PIC9k vector was subjected to double enzyme digestion reaction at the same time, and...

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Abstract

The invention discloses alpha-L-rhamnosidase as well as a preparation method and application of the alpha-L-rhamnosidase, in particular to an amino acid sequence of novel alpha-L-rhamnosidase, a nucleotide sequence for coding the amino acid sequence, a recombinant vector and a recombinant host containing the nucleotide sequence, a method for preparing the [alpha]-L-rhamnosidase by using the recombinant host and application of the [alpha]-L-rhamnosidase in baohuoside I production. The [alpha]-L-rhamnosidase provided by the invention has the advantages of high catalytic efficiency, good thermal stability, high specificity and the like. Epimedin C is subjected to rapid catalytic hydrolysis through glucosidase, then rhamnosidic bonds on the outer side of the C3 position are further subjected to catalytic hydrolysis through [alpha]-L-rhamnosidase provided by the invention to generate baohuoside I, the substrate conversion rate reaches up to 99.5%, the conversion efficiency is high, no by-product is generated, the steps are simple, the reaction time is short, pollution is small, and the method is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotransformation, and specifically relates to an alpha-L-rhamnosidase, its preparation method and application. Background technique [0002] Epimedium, also known as Xianlingpi and Gangqian, has the functions of nourishing kidney yang, strengthening muscles and bones, and dispelling rheumatism. At present, there are as many as 40 varieties of Epimedium, including Epimedium vellus, Epimedium sagitta and Epimedium Korean. Epimedium mainly contains major flavonoid glycosides such as epimedin A, epimedin B, epimedin C and icariin, of which icariin accounts for about 10%; epimedin A and B together account for than 10%; while Epimedin C ( figure 1 ) is the flavonoid saponin with the highest content in Epimedium, accounting for about 70%, and its C 3 position and C 7 The positions bear two rhamnosyls and one glucosyl, respectively. [0003] Baohuoside I ( figure 1 ), also known as Icariside ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P19/60C12P19/14C12R1/84C12R1/645
CPCC12N9/2402C12N15/815C12P19/60C12P19/14C12Y302/0104
Inventor 袁其朋程磊雨
Owner BEIJING UNIV OF CHEM TECH