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Alpha-cyclodextrin glucosyl transferase gene clone and expression

A glucosyl and cyclodextrin technology, which is applied in the fields of enzyme genetic engineering and enzyme engineering, can solve the problems of unfavorable recombinant α-CGT enzyme recovery and use, and achieve the effect of high thermal stability

Inactive Publication Date: 2008-10-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous reports, recombinant α-CGTase is generally localized in Escherichia coli to form inclusion bodies or exist in the periplasm, which is not conducive to the recovery and use of recombinant α-CGTase

Method used

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  • Alpha-cyclodextrin glucosyl transferase gene clone and expression
  • Alpha-cyclodextrin glucosyl transferase gene clone and expression
  • Alpha-cyclodextrin glucosyl transferase gene clone and expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example illustrates the extraction of total DNA from Paenibacillus macerans JFB05-01.

[0046] The P.macerans JFB05-01 strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 2 days, collected by centrifugation at 10,000rpm, washed with sterile water, and the collected precipitate was suspended in 500μL Add 15 μL of lysozyme to Tris-EDTA (trishydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer, incubate at 37°C for 30 minutes, then add 5 μL of RNase, incubate at 37°C for 30 minutes, add 30 μL of 10% SDS (dodecane sodium sulfate) and 15 μL proteinase K, incubated at 37°C for 60 min, added 100 μL NaCl (5M) and 80 μL CTAB (cetyltrimethylammonium bromide), incubated at 65°C for 20 min, and incubated with 700 μL of phenol: chloroform: iso Extract with a mixed solvent of amyl alcohol at a volume ratio of 25:24:1, centrifuge at 10,000 rpm, extract the supernatant with 700 μL of chloroform:isoamyl alcohol at a volume ratio ...

Embodiment 2

[0048] This example illustrates the cloning procedure for the gene encoding α-CGTase.

[0049] Using the total DNA of P.macerans JFB05-01 as a template, the following nucleotide sequences were used as primers, and the restriction sites Nco I and EcoR I were underlined to amplify the cgt gene by PCR.

[0050] Primer 1: 5'-CCATATgT CCATgg ATTCACCCgATACgAgC-3'

[0051] Primer 2: 5'-CTCgAgAg AATTC ggATTTTgCCAgTCCAC-3'

[0052] The PCR reaction was carried out in a 50 μL system: 10 μL of 5×PCR buffer, 4 μL of 2.5 mmol / L dNTPs, 1 μL of 10 μmol / L upstream and downstream primers, 1 μL of template DNA, 0.5 μL of Taq enzyme, and double distilled water to make the total system 50 μL;

[0053] The reaction conditions were 4 minutes of denaturation at 94°C, followed by 1 minute of denaturation at 94°C, 1 minute of annealing at 55°C, and 2 minutes of extension at 72°C for a total of 35 cycles, followed by 10 minutes of extension at 72°C. A PCR fragment of about 2100bp was amplified and...

Embodiment 3

[0055] This example illustrates the modification procedure of the cgt gene.

[0056] Since there is an Nco I restriction site inside the target gene, and the two ends of the gene are respectively Nco I and EcoR I sites, primers were designed to remove the mutation of the Nco I site to facilitate the cloning and expression in the next step. Use the pMD18-T simple vector connected to the cgt gene as a template for site-directed mutagenesis PCR, and design primers:

[0057] Primer 3: 5'-CAATgTgggTCCC ACA ATgggCCAgCC-3'

[0058] Primer 4: 5'-ggCTggCCCAT TgT gggACCCACATTg-3'

[0059] The PCR reaction was carried out in a 50 μL system, 5×PCR buffer 10 μL, 2.5 mmol / L dNTPs 4 μL, 10 μmol / L upstream and downstream primers 1 μL, template cgt / pMD18-T simple 1 μL, Taq enzyme 0.5 μL, add double distilled water to Total system 50μL;

[0060] The reaction conditions were denaturation at 94°C for 4 minutes, followed by denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, ...

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Abstract

The invention relates to the clone and the expression of alpha-cyclodextrin glycosyltransferase (alpha-CGTase), which belong to the field of enzyme gene engineering and enzyme engineering. The cgt gene expression method comprises the following steps: obtaining cgt gene SEQ ID NO:1 from total DNA of Peanibacillus macerans JFB05-01; selecting plasmid pET20b(+) as the expression vector of cgt gene; and selecting E.coli BL21(DE3) as the expression host to achieve high-efficiency extracellular expression of the cgt gene, wherein the cgt gene has 2,061 nucleotides and 687 coded amino acids; prokaryotic expression plasmid is constructed; and alpha-CGTase is expressed by transformed E.coli. Recombinase has cyclization activity and can transform starch and relevant matrix into cyclodextrin. The recombined alpha-CGTase has an optimal temperature of 40 to 45 DEG C and an optimal pH value of 5.5, and has higher thermal stability below 40 DEG C and poor thermal stability above 50 DEG C. The alpha-CGTase meets the requirement for industrial application such as food and medicines, and can be used for industrial production of alpha-cyclodextrin and beta-cyclodextrin.

Description

technical field [0001] The invention relates to the cloning and expression of an α-cyclodextrin glucosyltransferase (abbreviated as α-CGTase) gene, and the invention belongs to the fields of enzyme gene engineering and enzyme engineering. Specifically, it relates to a DNA sequence encoding a Peanibacillus macerans JFB05-01α-CGT enzyme and its expression. Background technique [0002] CGTase is a multifunctional enzyme that catalyzes four different reactions: three transglycosylation reactions (disproportionation, cyclization, and coupling) and hydrolysis. CGTase can utilize starch to produce cyclodextrin through cyclization reaction, which is the basis of its industrial application. In addition to the production of cyclodextrins by cyclization reactions, CGTases have recently been used to catalyze coupling and disproportionation reactions to transfer oligosaccharides from donors such as starch or cyclodextrins to various acceptor molecules to obtain various special oligosac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70
Inventor 陈坚吴敬李兆丰李彬成成
Owner JIANGNAN UNIV
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