Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof

A technology of genetically engineered bacteria and isoamylase, applied in the field of enzyme engineering, can solve the problems of too expensive raw materials, substrate concentration, long reaction cycle, and difficulty in industrialized production, and achieve the effect of high enzyme activity

Active Publication Date: 2012-07-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these processes have serious problems such as too expensive raw materials or too low subst

Method used

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  • Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof
  • Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof
  • Acidic heat-resisting isoamylase genetic engineering bacterium and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: the construction of genetically engineered bacteria

[0021] 1. According to the gene sequence of Tfu_1891 registered on NCBI (Genbank No. NC_007333.1), the thermostable isoamylase gene sequence was synthesized by chemical total synthesis method, cloned into the pMD18-T simple vector (commercial tool vector), and the ligated product was transformed Escherichia coli JM109, the transformation product was spread on LB plates containing 100 mg / L ampicillin. After culturing overnight at 37°C, colonies were selected and inserted into LB liquid medium. After 8-10 hours, the plasmid was extracted and named as Tfu_1891 / pMD18-T simple. The plasmid was sequenced. The results showed that the inserted fragment was a 2124bp DNA fragment encoding a protein with 708 amino acids.

[0022] 2. The plasmid used to construct the expression vector in Escherichia coli is pT7-7 with a T7 promoter. The pT7-7 plasmid and Tfu_1891 / pMD18-T simple were digested with Nde I and HindII...

Embodiment 2

[0024] Embodiment 2: fermentation produces isoamylase

[0025] Transfer the glycerol tube strain to LB medium for 37°C liquid culture overnight, and then insert TB (glycerol 5g / L, peptone 12g / L, yeast extract 24g / L, K 2 HPO 4 12.54g / L, KH 2 PO 4 2.31g / L) fermentation liquid medium, cultured at 30°C until OD reached 0.6, then induced with a final concentration of 0.01μM isopropylthio-β-D-galactoside, and cultured at constant temperature or lowered to 25°C for 36 to 48 hours, Centrifuge the bacteria, suspend the cells with pH5.50.1M phosphate-citrate buffer, ultrasonically disrupt, measure the activity of isoamylase in the supernatant after centrifugation, and the fermentation activity of recombinant isoamylase reaches 3185U / mL.

Embodiment 3

[0026] Example 3: Purification and Enzymatic Properties of Recombinant Isoamylase

[0027] The sonicated supernatant was centrifuged at 40,000×g for 20 min at 4°C to remove cell debris. Add 20% solid ammonium sulfate to the supernatant for salting out overnight, centrifuge at 40,000×g for 20 minutes at 4°C, dissolve the precipitate with an appropriate amount of pH 5.5, 20mM phosphoric acid-citric acid buffer, and filter it through a 0.4μm membrane to make a sample sample. After purified by DEAE-Sepharose anion exchange resin, the sample was added to Sephadex G50 for purification to obtain electrophoretic pure isoamylase. figure 1 .

[0028]The recombinant isoamylase was tested for hydrolysis of different substrates, and it was found that it could hydrolyze waxy corn amylopectin, but not glycogen and pullulan. The results showed that the gene encoding recombinant isoamylase was highly expressed. The optimum temperature for acid thermostable isoamylase is 50°C ( figure 2 ),...

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Abstract

The invention relates to an acidic heat-resisting isoamylase genetic engineering bacterium and an application thereof, belonging to the field of enzyme engineering. According to the invention, a Tfu_1891 encoding gene sequence of isoamylase is obtained by chemical synthesis; culture is carried out in an industrial fermentation culture medium by taking pT7-7 as an expression vector and E.coli BL21 (DE3) as an expression host; and by virtue of inductive recombination, the fermentation vigor of the isoamylase reaches 3185U/mL. The recombinase has hydrolysis activity of a starch alpha-1,6 glucoside bond; the optimum temperature is 40-50 DEG C, and the optimum pH value is 5.5; after heat preservation is carried out for 30 hours at the temperature of 50 DEG C, the enzyme activity of the recombinase still maintains more than 50%; the recombinase has starch debranching activity and can hydrolyze branched starch in starch so as to form amylose; and the recombinase is combined with alpha-cyclodextrin glucosyltransferase to be used, thereby obviously improving the conversion rate of cyclodextrin and being suitable for industrial production of cyclodextrin.

Description

technical field [0001] The invention relates to an acid heat-resistant isoamylase genetically engineered bacterium and application thereof, belonging to the field of enzyme engineering. Background technique [0002] Isoamylase (E.C.3.2.1.68) is a class of starch debranching enzymes capable of hydrolyzing α-1,6 glucosidic bonds, which can hydrolyze glycogen, amylopectin, α-limit dextrin and β-limit dextrin , The hydrolyzate is linear maltooligosaccharide. Although both plants and microorganisms can produce isoamylases, commercial isoamylases are generally produced by microorganisms due to their advantages of rapid growth, simple operation, and low cost. The most studied isoamylase-producing strains are Pseudomonas (Pseudomonas.sp.), followed by other bacteria and fungi. Isoamylases from different sources have different substrate specificity, hydrolyzate, optimum pH, optimum temperature and so on. Isoamylase is mainly used to hydrolyze starch to produce food additives, such...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/56C12N15/63C12P19/18C12P19/16C12R1/19
Inventor 吴敬陈晟段绪果陈坚
Owner JIANGNAN UNIV
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