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Sucrose isomerase gene and high-efficiency expression method thereof

A technology of molasses and isomaltulose, which is applied in the fields of genetic engineering and enzyme engineering, and achieves the effects of short induction time, high expression, convenient and cheap use

Inactive Publication Date: 2012-09-05
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In China, the cloning and expression of SIase has been rarely reported so far, and the limited research in China is still mainly devoted to improving the enzyme production of the original strain

Method used

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  • Sucrose isomerase gene and high-efficiency expression method thereof
  • Sucrose isomerase gene and high-efficiency expression method thereof
  • Sucrose isomerase gene and high-efficiency expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Extraction of total DNA of Erwinia rhapontici NX-5.

[0032] E.rhapontici NX-5 strain (CCTCC NO: 2222) was cultured in SB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 12h, centrifuged at 8000r / min to collect the bacteria, sterile water Wash, collect the precipitate and suspend it in 500μL Tris-EDTA (Tris-hydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer, add 5μL RNase, incubate at 37°C for 30min, add 30μL 10% SDS (sodium dodecyl sulfate) And 15μL of proteinase K, incubate at 37℃ for 60min, add 100μL of 5M NaCl solution and 80μL CTAB (hexadecyltrimethylammonium bromide), incubate at 65℃ for 20min, use 700μL of phenol: chloroform: isoamyl alcohol volume The ratio of 25:24:1 mixed solution extraction, 10000r / min centrifugation, the supernatant with 700μL chloroform: isoamyl alcohol volume ratio 24:1 mixed solvent extraction, 10000r / min centrifugation, the supernatant and 1400μL of ice Mix with amyl alcohol, precipitate at -20°C...

Embodiment 2

[0033] Example 2: Cloning of SIase encoding gene.

[0034] Using the E.rhapontici NX-5 total DNA obtained in Example 1 as a template, the following nucleotide sequences were used as primers:

[0035] Primer 1: TT AAGCTT CCATGG ATTCTCAAGGATT, respectively introduce HindIII, Nco I restriction sites (underlined part).

[0036] Primer 2: GTAAATATTTGAATTAGGC GAGCTC CCTAGG TT, respectively introduce BamH I and Xho I double restriction sites (underlined part).

[0037] PCR reaction was carried out in a 20μL system: 10×PCR buffer 2μL, 2.5mmol / L dNTP 2μL, 10μmol / L primer 1 and primer 2 each 2μL, template DNA 2μL, 25mmol / LMgCl 2 2μL, TaqDNA polymerase 1μL, add double distilled water to 20μL.

[0038] PCR reaction conditions: denaturation at 95°C for 3min, then start cycling, (94°C 30s; 45°C 30s; 72°C 2min; 72°C 5min), a total of 25 cycles, amplify a 1800bp PCR fragment, cut the gel and recover the fragment After double digestion with HindIII and BamH I, the DNA fragment purification kit wa...

Embodiment 3

[0039] Example 3: Construction of pal I gene on an expression vector.

[0040] The plasmid used to construct the expression vector is pET22b(+), with pelB signal peptide and His-tag tag. The pET22b(+) plasmid and pal I gene were digested with Xho I and Nco I by double restriction enzymes. After the gel was cut to recover pal I, the pal I was ligated overnight at 16°C with T4 ligase. The ligation product was transformed into E.coli BL21(DE3) competent cells. Incubate overnight at 37°C, select the transformants for liquid culture in 100 μg / mL ampicillin LB, and then extract the plasmid to obtain the enriched pal I / pET22b(+) plasmid ( figure 1 ).

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Abstract

The invention discloses a sucrose isomerase gene, namely a pal I gene, which has the nucleotide sequence shown as SEQ ID NO:1. The invention also discloses a coding protein for the gene, namely an SIase enzyme. The invention also discloses an expression vector and a host cell containing the gene, and a high-efficiency expression method for the gene. A recombinant strain containing the sucrose isomerase gene has isomerism activity and can transform sucrose into isomaltulose. The recombinant strain has the advantages of high stability, high catalytic efficiency, obvious improvement of product specificity, and application to industrial production of the isomaltulose.

Description

Technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a sucrose isomerase gene and a high-efficiency expression method thereof. Background technique [0002] Sucrose isomerase (SIase) is a key enzyme for producing functional sweeteners isomaltulose (alcohol) and trehalulose. Isomalt is a kind of functional sugar alcohol emerging in recent years. It is prepared by hydrogenation of isomaltulose through nickel catalyst. In addition to the common characteristics of general sugar alcohols, it also has extremely low hygroscopicity and pure taste that cannot be compared with functional sugar alcohols such as xylitol and maltitol. Because of its unique properties, it has been welcomed by the food industry to produce sugar-free sweet foods. Especially in recent years, as people pay attention to their own health, the production, development and utilization of isomalt has received extensive attention. It has b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/63C12N1/21C12P19/12C12R1/19
Inventor 徐虹李莎任贲蔡恒汪晨
Owner NANJING TECH UNIV
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