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Efficient preparation method and gene engineering bacteria of sucrose isomerase

A sucrose isomerase and a construction method technology are applied in the field of genetically engineered bacteria with high sucrose isomerase production and high-efficiency production of sucrose isomerase, and can solve the problems of long transformation time, low cell enzyme activity, low cell yield and the like , to achieve the effect of increased secretion, efficient secretion and expression, and increased expression

Active Publication Date: 2016-01-20
森大(天津)生物科技有限公司
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  • Description
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Problems solved by technology

However, most of the wild strains that have been reported so far have problems such as low cell yield, low cell enzyme activity, and poor tolerance under high-concentration substrates, resulting in low conversion rates and long conversion times.

Method used

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  • Efficient preparation method and gene engineering bacteria of sucrose isomerase
  • Efficient preparation method and gene engineering bacteria of sucrose isomerase
  • Efficient preparation method and gene engineering bacteria of sucrose isomerase

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Codon optimization of embodiment 1 sucrose isomerase coding gene

[0031]Using the genomic DNA of PantoeadispersaUQ68J as a template, primers were designed according to the sequence of the sucrose isomerase gene sim (GenBank accession number: AY223549.1) of P.dispersaUQ68J in the NCBI database, and the sim gene ( Nucleotide sequence SEQIDNO: 1. PCR amplification system and reaction conditions reference HSDNA Polymerase instruction sheet (TaKaRa). The PCR product was cloned into the XbaI and KpnI sites of the expression vector pHY-WZX (DandanNiuandZhengxiangWang.JIndMicrobiolBiotechnol, 2007, 34:357-362) to obtain the recombinant expression plasmid pHY-sim( figure 1 a). Based on pHY-sim, through the website http: / / genomes.urv.es / OPTIMIZER / , optimize the codon composition of the gene encoding sucrose isomerase, and through the whole gene synthesis technology (Niu Dandan et al. Applied and Environmental Biology Technical Journal, 2007, 13 (4): 515-518), obtained codon-...

Embodiment 2

[0032] Example 2 Screening of Signal Peptides for Efficient Secretion and Expression of Sucrose Isomerase

[0033] 173 signal peptide sequences were searched from the signal peptide library of homologous proteins of Bacillus licheniformis, and expression vectors fused with these signal peptides and sucrose isomerase were constructed, and the secretion effect of sucrose isomerase was improved to varying degrees from the clones by screening The signal peptide specifically comprises the following steps:

[0034] 1. Construction of plasmid pHY-signalpeptidedatabase-sim2

[0035] Bacillus licheniformis (Bacillus licheniformis) CBBD302 (Dandan Niu, et al. Microbial Cell Factories, 2009, 8:58) genomic DNA (bacterial genomic DNA extraction kit) was extracted, and the selected signal peptide DNA was amplified by PCR with corresponding primers. At the same time, using the plasmid pHY-sim as a template, the sequence of the promoter (Promoter) was amplified by PCR. The two fragments wer...

Embodiment 3

[0061] The synthesis level of sucrose isomerase in the recombinant bacillus licheniformis of embodiment 3

[0062] The recombinant expression plasmids pHY-sim, pHY-sim2 and pHY-sYbdN-sim2 obtained in the above Examples 1 and 2 were transformed into the host cell Bacillus licheniformis CBBD302 by electroporation, and the transformants were screened on a selective plate , respectively named as CBBD302-1, CBBD302-2 and CBBD302-sYbdN. Fermentation was carried out in 250 mL Erlenmeyer flasks containing 50 mL of culture medium. Fermentation is carried out in a fermentation medium (yeast extract 0.5-1.5%, peptone 1-4%, glucose 10-20%, the rest is water, pH 7.0), at 42°C and 220rpm, the fermentation time is 120h, and the fermentation time is measured. The enzyme activity of sucrose isomerase in the solution is summarized in Table 2. Compared with the original strain CBBD302-1, the synthetic level of sucrose isomerase in the recombinant strain CBBD302-2 was increased by 76%. The lev...

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Abstract

The invention discloses gene engineering bacteria with a high yield of sucrose isomerase, and a method for efficiently producing the sucrose isomerase, and belongs to the technical field of gene engineering and fermentation engineering. According to the method, codon and signal peptide optimization is adopted to ensure that the expression quantity of a sucrose isomerase gene in bacillus licheniformis is improved by more than or equal to 3 times compared with the original expression quantity. Finally, a growth medium and a fermentation process suitable for synthesis and secretion of an enzyme, and recovery and preparation of a product are established, so that the content of isomaltulose in the product reaches 100%. Therefore, a firm foundation is laid for the application of the sucrose isomerase in food and other industries, and a technical support is provided for efficient preparation of other industrial enzyme preparations.

Description

technical field [0001] The invention belongs to the field of genetic engineering and fermentation engineering, and in particular relates to a genetically engineered bacterium for high-production sucrose isomerase and a method for efficiently producing sucrose isomerase. Background technique [0002] Isomaltulose (6-O-α-D-glucopyranosyl-D-fructose, Isomaltulose), also known as Palatinose, exists in honey and sugarcane juice at trace levels in nature. It is an isomer with sucrose, not only has the physical properties and taste of sucrose, but also has the characteristics of low sweetness, acid hydrolysis resistance, caries prevention and low hygroscopicity, so it has been favored by the food industry for sugar-free sweet food Welcome, has gradually been used as a substitute for sucrose. [0003] Sucrose isomerase (Sucroseisomerase, SIA, E.C.5.4.99.11) is widely used in the industrial production of isomaltulose. The enzyme catalyzes the isomerization of sucrose to produce iso...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12N15/75C12N1/21C12P19/24C12P19/12C12R1/10
Inventor 王正祥路福平
Owner 森大(天津)生物科技有限公司
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